Method for detection of drug-induced mutations in the reverse transcriptase gene

ABSTRACT

The present invention relates to a method for the rapid and reliable detection of drug-induced mutations in the reverse transcriptase gene allowing the simultaneous characterization of a range of codons involved in drug resistance using specific sets of probes optimized to function together in a reverse-hybridization assay. More particularly, the present invention relates to a method for determining the susceptibility to antiviral drugs of HIV strains present in a biological sample, comprising: (i) if need be releasing, isolating or concentrating the polynucleic acids present in the sample; (ii) if need be amplifying the relevant part of the reverse transcriptase genes present in said sample with at least one suitable primer pair; (iii) hybridizing the polynucleic acids of step (i) or (ii) with at least two RT gene probes hybridizing specifically to one or more target sequences with said probes being applied to known locations on a solid support and with said probes being capable of simultaneously hybridizing to their respective target regions under appropriate hybridization and wash conditions allowing the detection of homologous targets, or said probes hybridizing specifically with a sequence complementary to any of said target sequences, or a sequence wherein T is replaced by U; (iv) detecting the hybrids formed in step (iii); (v) inferring the nucleotide sequence at the codons of interest and/or the amino acids of the codons of interest and/or antiviral drug resistance spectrum, and possibly the type of HIV isolates involved from the differential hybridization signal(s) obtained in step (iv).

BACKGROUND OF THE INVENTION

The present invention relates to the field of HIV diagnosis. Moreparticularly, the present invention relates to the field of diagnosingthe susceptibility of an HIV sample to antiviral drugs used to treat HIVinfection.

1. Field of the Invention

The present invention relates to a method for the rapid and reliabledetection of drug-induced mutations in the HIV reverse transcriptasegene allowing the simultaneous characterization of a range of codonsinvolved in drug resistance using specific sets of probes optimized tofunction together in a reverse-hybridisation assay.

2. Description of the Related Art

During the treatment of human immunodeficiency virus (HIV) type 1infected individuals with antiretroviral nucleoside analogs emergence ofresistance against these drugs has been observed. The mechanismresponsible for the resistance is not fully understood, since theappearance of a resistant virus in not always correlated with clinicaldeterioration (Boucher et al. 1992). Amongst the reverse transcriptase(RT) inhibitors, the nucleoside analogs 3'-azido-2',3'-dideoxyThymidine(AZT, Zidovudine), 2',3'-dideoxyInosine (ddI), 2',3'-dideoxyCytidine(ddC), (-)-β-L-2',3'-dideoxy-3'-thioCytidine (3TC),2',3'-didehydro-3'deoxyThymidine (D4T) and(-)-2'3'-dideoxy-5-fluoro-3'-thiacytidine (FTC) are the most important,since they show a favourable ratio of toxicity for the host versusefficacy as antiviral. All these compounds act in a similar way, namelythey serve, after intracellular phosphorylation, as chain terminators ofthe RT reaction. Upon prolonged treatment with these nucleoside analogs,accumulation of mutations in the viral reverse transcriptase gene (RT)occur, thereby escaping the inhibitory effect of the antivirals. Themost important mutations induced by the above compounds and leading togradually increasing resistance were found at amino acid (aa) positions41 (M to L), 69 (T to D), 70 (K to R), 74 (L to V), 181 (Y to C), 184 (Mto V) and 215 (T to Y or F) (Schinazi et al., 1994). Mutations at aa 65,67, 75 and 219 have also been reported but these were only showing aminor decrease in sensitivity. More recently, multi-drug-resistant HIV-1strains were described showing aa changes at codon 62, 75, 77, 116, and151 (Iversen et al., 1996). In general, these aa changes are theconsequence of single point mutations at the first or second codonletter, but in the case of T69D (ACT to GAT), T215Y (ACC to TAC) andT215F (ACC to TTC), two nucleotide mutations are necessary. Whether inthese cases the single nucleotide mutation intermediates exist, and ifthey show any importance in the mechanism for acquiring resistance is asyet not reported. Third letter variations are in general not leading toan amino acid change, and are therefore seen as natural polymorphisms.

The regime for an efficient antiviral treatment is not clear at all. Theappearance of one or several of these mutations during antiviraltreatment need to be interpreted in conjunction with the virus load andthe amount of CD4 cells. Indeed, since it has been shown that the effectof AZT resistance mutations can be suppressed after the appearance ofthe 3TC induced M184V mutation, it is clear that disease progression ismultifactorial. The influence of other simultaneous occuring mutationsunder different combination therapies with respect to the outcome andresistance of the virus has not yet been analysed systematically. Inorder to get a better insight into the mechanisms of resistance and HIVbiology, it is necessary to analyse follow-up plasma samples ofantiviral treated patients for these mutational events together with thesimultaneous occuring changes of virus titre and CD4 cells.

BRIEF SUMMARY OF THE INVENTION

It is an aim of the present invention to develop a rapid and reliabledetection method for determination of the antiviral drug resistance ofviruses which contain reverse transcriptase genes such as HIVretroviruses and Hepadnaviridae present in a biological sample.

More particularly it is an aim of the present invention to provide agenotyping assay allowing the detection of the different HIV RT genewild type and mutation codons involved in the antiviral resistance inone single experiment.

It is also an aim of the present invention to provide an HIV RTgenotyping assay or method which allows to infer the nucleotide sequenceat codons of interest and/or the amino acids at the codons of interestand/or the antiviral drug resistance spectrum, and possibly also inferthe HIV type or subtype isolate involved.

Even more particularly it is an aim of the present invention to providea genotyping assay allowing the detection of the different HIV RT genepolymorphisms representing wild-type and mutation codons in one singleexperimental setup.

It is another aim of the present invention to select particular probesable to discriminate wild-type HIV RT sequences from mutated orpolymorphic HIV RT sequences conferring resistance to one or moreantiviral drugs, such as AZT, ddI, ddC, 3TC or FTC, D4T or others.

It is more particularly an aim of the present invention to selectparticular probes able to discriminate wild-type HIV RT sequences frommutated or polymorphic HIV RT sequences conferring resistance to AZT.

It is more particularly an aim of the present invention to selectparticular probes able to discriminate wild-type HIV RT sequences frommutated HIV RT sequences conferring resistance to ddI.

It is more particularly an aim of the present invention to selectparticular probes able to discriminate wild-type HIV RT sequences frommutated HIV RT sequences conferring resistance to ddC.

It is more particularly an aim of the present invention to selectparticular probes able to discriminate wild-type HIV RT sequences frommutated HIV RT sequences conferring resistance to 3TC.

It is more particularly an aim of the present invention to selectparticular probes able to discriminate wild-type HIV RT sequences frommutated HIV RT sequences conferring resistance to D4T.

It is more particularly an aim of the present invention to selectparticular probes able to discriminate wild-type HIV RT sequences frommutated HIV RT sequences conferring resistance to FTC.

It is more particularly an aim of the present invention to selectparticular probes able to discriminate wild-type HIV RT sequences frommutated HIV RT sequences conferring resistance to multiple nucleosideanalogues (i.e. multidrug resistance).

It is more particularly an aim of the present invention to selectparticular probes able to discriminate wild-type HIV RT sequences frommutated HIV RT sequences conferring resistance to nevirapine.

It is more particularly an aim of the present invention to selectparticular probes able to discriminate wild-type HIV RT from mutated HIVRT sequences involving at least one of amino acid positions 41 (M to L),50 (I to T), 67 (D to N), 69 (T to D), 70 (K to R), 74 (L to V), 75 (Vto T), 151 (Q to M or L), 181 (Y to C), 184 (M to V), 215 (T to Y or F)and 219 (K to Q or E) of the viral reverse transcriptase (RT) gene.

It is particularly an aim of the present invention to select aparticular set of probes, able to discriminate wild-type HIV RTsequences from mutated HIV RT sequences conferring resistance to any ofthe antiviral drugs defined above with this particular set of probesbeing used in a reverse hybridisation assay.

It is moreover an aim of the present invention to combine a set ofselected probes able to discriminate wild-type HIV RT sequences frommutated HIV RT sequences conferring resistance to antiviral drugs withanother set of selected probes able to identify the HIV isolate, type orsubtype present in the biological sample, whereby all probes can be usedunder the same hybridisation and wash-conditions.

It is also an aim of the present invention to select primers enablingthe amplification of the gene fragment(s) determining the antiviral drugresistance trait of interest.

The present invention also aims at diagnostic kits comprising saidprobes useful for developing such a genotyping assay.

All the aims of the present invention have been met by the followingspecific embodiments.

The present invention relates more particularly to a method fordetermining the susceptibility to antiviral drugs of an HIV retroviruspresent in a biological sample, comprising:

(i) if need be releasing, isolating or concentrating the polynucleicacids present in the sample;

(ii) if need be amplifying the relevant part of the reversetranscriptase genes present in said sample with at least one suitableprimer pair;

(iii) hybridizing the polynucleic acids of step (i) or (ii) with atleast two RT gene probes hybridizing specifically to at least one targetsequence as mentioned in any of FIG. 1 and tables 1, 2 or 4, with saidprobes being applied to known locations of a solid support and with saidprobes being capable of simultaneously hybridizing to their respectivetarget regions under appropiate hybridization and wash conditionsallowing the detection of homologous targets, or with said probeshybridizing specifically with a sequence complementary to any of saidtarget sequences, or a sequence wherein T in said target sequence isreplaced by U;

(iv) detecting the hybrids formed in step (iii);

(v) and in most cases, inferring the nucleotide sequence at the codonsof interest and/or the amino acids at the codons of interest and/or theantiviral drug resistance spectrum, and possibly the type of HIVisolates involved from the differential hybridization signal(s) obtainedin step (iv).

The relevant part of the RT gene refers to the regions in the RT geneharboring mutations causing resistance to antiviral drugs as describedabove and is particularly comprised between codons 1 and 241, and moreparticularly between codons 29 and 220 of the RT gene.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Natural and drug induced variability in the vicinity of codons41, 50, 67-70, 74-75, 150, 181-184, 215 and 219 of the HIV RT gene. Themost frequently observed wild-type sequence is shown in the top line.Naturally occuring variations are indicated below. Drug-induced variantsare indicated in bold italics.

FIG. 2A. Reactivities of the selected probes for codon 41 immobilized onLiPA strips with reference material. The position of each probe on themembrane strip is shown at the right of each panel. The sequence of therelevant part of the selected probes is given in Table 4. Each strip isincubated with a biotinylated PCR fragment from the reference panel. Thereference panel accession numbers are indicated in Table 4. For severalprobes multiple reference panel possibilities are available, but onlyone relevant accession number given each time. *: False positivereactivities. On top of the strips, the amino acids at the relevantcodon, as derived from the probe reactivity, is indicated.

FIG. 2B. Reactivities of the selected probes for codons 69-70immobilized on LiPA strips with reference material. The position of eachprobe on the membrane strip is shown at the right of each panel. Thesequence of the relevant part of the selected probes is given in Table4. Each strip is incubated with a biotinylated PCR fragment from thereference panel. The reference panel accession numbers are indicated inTable 4. For several probes multiple reference panel possibilities areavailable, but only one relevant accession number given each time. *:False positive reactivities. On top of the strips, the amino acids atthe relevant codon, as derived from the probe reactivity, is indicated.

FIG. 2C. Reactivities of the selected probes for codons 74-75immobilized on LiPA strips with reference material. The position of eachprobe on the membrane strip is shown at the right of each panel. Thesequence of the relevant part of the selected probes is given in Table4. Each strip is incubated with a biotinylated PCR fragment from thereference panel. The reference panel accession numbers are indicated inTable 4. For several probes multiple reference panel possibilities areavailable, but only one relevant accession number given each time. Ontop of the strips, the amino acids at the relevant codon, as derivedfrom the probe reactivity, is indicated.

FIG. 2D. Reactivities of the selected probes for codon 184 immobilizedon LiPA strips with reference material. The position of each probe onthe membrane strip is shown at the right of each panel. The sequence ofthe relevant part of the selected probes is given in Table 4. Each stripis incubated with a biotinylated PCR fragment from the reference panel.The reference panel accession numbers are indicated in Table 4. Forseveral probes multiple reference panel possibilities are available, butonly one relevant accession number given each time. On top of thestrips, the amino acids at the relevant codon, as derived from the probereactivity, is indicated.

FIG. 2E. Reactivities of the selected probes for codon 215 immobilizedon LiPA strips with reference material. The position of each probe onthe membrane strip is shown at the right of each panel. The sequence ofthe relevant part of the selected probes is given in Table 4. Each stripis incubated with a biotinylated PCR fragment from the reference panel.The reference panel accession numbers are indicated in Table 4. Forseveral probes multiple reference panel possibilities are available, butonly one relevant accession number given each time. On top of thestrips, the amino acids at the relevant codon, as derived from the probereactivity, is indicated.

FIG. 2F. Reactivities of the selected probes for codon 219 immobilizedon LiPA strips with reference material. The position of each probe onthe membrane strip is shown at the right of each panel. The sequence ofthe relevant part of the selected probes is given in Table 4. Each stripis incubated with a biotinylated PCR fragment from the reference panel.The reference panel accession numbers are indicated in Table 4. Forseveral probes multiple reference panel possibilities are available, butonly one relevant accession number given each time. On top of thestrips, the amino acids at the relevant codon, as derived from the probereactivity, is indicated.

FIG. 3. Clinical and virological features detectable in three patientfollow-up samples. All three patients were infected with a HIV-1 strainshowing the M41-T69-K70-L74-V75-M184-F214-T215-K219 genotype (wild typepattern). Top: Fluctuations between plasma HIV RNA copy numbers (▪) andCD4 cell count (x) are given in function of time. The differenttreatment regimens and the period of treatment is indicated on top.Middle: Changes that appeared during the treatment period and that couldbe scored by means of the LiPA probes are indicated, for patient 91007at codon 41 and 215; for patient 94013 at codon 184; for patient 92021at codon 70, 214, 215, 219. Bottem: Corresponding LiPA strips for asubset of the aa changes are shown. LiPA probes are indicated on theleft, the aa interpretation is indicated at the right of each panel.

FIG. 4. Reactivities of the selected probes for codons 151 and 181 onLiPA strips with reference material. The position of each probe on themembrane strip is shown at the right of each panel. The sequence of therelevant part of the selected probes is given in Table 3. LiPA stripswere incubated with sequence-confirmed PCR fragments, extracted andamplified from: a wild-type HIV-1 isolate (strip 1), a wild-type isolatewith a polymorphism at codon 151 (strip 2) or 149 (strip 3), amulti-drug resistant HIV-1 isolate (strip 4) with no information aboutcodon 181 and a non-nucleoside analogue treated HIV-1 isolate whichremained wild-type at codon 151 (strip5).

DETAILED DESCRIPTION OF THE INVENTION

According to a preferred embodiment of the present invention, step (iii)is performed using a set of at least 2, preferably at least 3, morepreferably at least 4 and most preferably at least 5 probes meticulouslydesigned as such that they show the desired hybridization results, whenused in a reverse hybridisation assay format, more particularly underthe same hybridization and wash conditions.

According to a preferred embodiment, the present invention relates to aset of at least 2 probes each targetting one or more of the nucleosideRT inhibitor induced nucieotide changes or target sequences includingsuch a nucleotide change as indicated in any of FIG. 1 or Tables 1, 2 or4. The numbering of HIV-1 RT gene encoded amino acids is as generallyaccepted in literature.

More prefererably, the present invention relates to a set of two or moreprobes each targetting two, three, four, five or more differentnucleoside RT inhibitor induced nucleotide changes as indicated in anyof FIG. 1 or Tables 1, 2 or 4.

More particularly, the present invention relates to a set of at least 2probes allowing the characterization of a wild-type, polymorphic ormutated codon at any one of the drug-induced mutation positionsrepresented in any of FIG. 1 or Tables 1 or 2 or at any one of thepolymorphic positions represented in Table 4.

Even more particularly, the present invention relates to a set of atleast 2 probes allowing the characterization of a wild-type, polymorphicor mutated codon at any of the positions represented in FIG. 1.

All the above mentioned sets of probes have as a common characteristicthat all the probes in said set are designed so that they can functiontogether in a reverse-hybridization assay, more particularly undersimilar hybridization and wash conditions.

A particularly preferred set of probes selected out of the probes withSEQ ID NO 1 to 161 of Table 3 is described in example 2.2 and isindicated in Table 4 and FIG. 2. The particularly selected probes arealso indicated in Table 3.

A particularly preferred embodiment of the present invention is a methodfor determining the susceptibility to antiviral drugs of an HIV isolatesin a sample using a set of probes as defined above, wherein said set ofprobes is characterized as being chosen such that for a given mutationdisclosed in any of FIG. 1, or Tables 1, 2 or 4 the following probes areincluded in said set:

at least one probe for detecting the presence of drug induced mutationat said position;

at least one probe for detecting the presence of a wild-type sequence atsaid position;

preferably also (an) additional probe(s) for detecting wild-typepolymorphisms at positions surrounding the mutation position.

Inclusion of the latter two types of probes greatly contributes toincreasing the sensitivity of said assays as demonstrated in theexamples section.

A particularly preferred set of probes in this respect is shown inTables 3 and 4 and FIGS. 2 and 3.

Selected sets of probes according to the present invention include atleast one probe, preferably at least two probes, characterizing thepresence of a drug-induced mutation in a codon position chosen from thefollowing list of codons susceptible to mutations in the HIV RT gene:41, 50, 67, 69, 70, 74, 75, 151, 181, 184, 215 or 219. Said probes beingcharacterized in that they can function in a method as set out above.

Also selected probes according to the present invention are probes whichallow to differentiate any of the nucleotide changes as represented inany of FIG. 1 or Tables 1, 2 or 4. Said probes being characterized inthat they can function in a method as set out above.

Also selected sets of probes for use in a method according to thepresent invention include at least one, preferably at least two (setsof) probes, with said probes characterizing the presence of adrug-induced mutation in two codon positions chosen from the followinglist of codon combinations, with said codons being susceptible tomutations in the HIV RT gene: 41 and/or 50; 41 and/or 67; 41 and/or 69;41 and/or 70; 41 and/or 74; 41 and/or 75; 41 and/or 151; 1 and/or 181;41 and/or 184; 41 and/or 215; 41 and/or 219; 50 and/or 67; 50 and/or 69;50 and/or 70; 50 and/or 74; 50 and/or 75; 50 and/or 75; 50 and/or 151;50 and/or 181; 50 and/or 184; 50 and/or 215; 50 and/or 219; 67 and/or69; 67 and/or 70; 67 and/or 74; 67 and/or 75; 67 and/or 151; 67 and/or181; 67 and/or 184; 67 and/or 215; 67 and/or 219; 69 and/or 70; 69and/or 74; 69 and/or 75; 69 and/or 151; 69 and/or 181; 69 and/or 184; 69and/or 215; 69 and/or 219; 70 and/or 74; 70 and/or 75; 70 and/or 151; 70and/or 181; 70 and/or 184; 70 and/or 215; 70 and/or 219; 74 and/or 75;74 and/or 151; 74 and/or 181; 74 and/or 184; 74 and/or 215; 74 and/or219; 75 and/or 151; 75 and/or 181; 75 and/or 184; 75 and/or 215; 75and/or 219; 151 and/or 181; 151 and/or 184; 151 and/or 215; 151 and/or219; 181 and/or 184; 181 and/or 215; 181 and/or 219; 184 and/or 215; 184and/or 219; 215 and/or 219.

Even more preferred selected sets of probes for use in a methodaccording to the present invention include in addition to the probesdefined above a third (set of) probe(s) characterizing the presence of athird drug-induced mutation at any of positions 41, 50, 67, 69, 70, 74,75, 151. 181, 184, 215 or 219, or particular combinations thereof.

Particularly preferred is also a set of probes which allows simultaneousdetection of antiviral resistance at codons 41, 50, 67, 69, 70, 74, 75,151, 181, 184 and 215, possibly also at codon 219.

An additional embodiment of the present invention includes at least oneprobe, preferably at least two probes, characterizing the presence of adrug-induced mutation in codon positions chosen from the list of codonssusceptible to mutations in the HIV RT gene as mentioned in any of Table1 or 2, such as at codons 65, 115, 150, 98, 100, 103, 106, 108, 188,190, 138, 199, 101, 179, 236, 238 or 233, with said probes formingpossibly part of a composition.

Particularly preferred embodiments of the invention thus include a setof probes for codon 41 comprising at least one, preferably at least two,probe(s) for targetting at least one, preferably at least two,nucleotide changes in any the following codons as represented in regionI in FIG. 1:

wild-type codon E40 (GAA) or polymorphic codon E40 (GAG), mutant codonL41 (TTG) or L41 (CTG) or wild-type codon M41 (ATG), wild-type codon E42(GAA) or polymorphic codon E42 (GAG), wild-type codon K43 (AAG) orpolymorphic codon K43 (AAA) or polymorphic E43 (GAA).

Particularly preferred embodiments of the invention thus include a setof probes for codon 50 comprising at least one, preferably at least two,probe(s) for targetting at least one, preferably at least two,nucleotide changes in any the following codons as represented in regionII in FIG. 1:

wild-type codon K49 (AAA) or polymorphic codon R49 (AGA), mutant codonsV50 (GTT) or T50 (ACG), wild-type codon I50 (ATT) or polymorphic codonI50 (ATC).

Particularly preferred embodiments of the invention thus include a setof probes for codons 67-70 comprising at least one, preferably at leasttwo, probe(s) for targetting at least one, preferably at least two,nucleotide changes in any of the following codons as represented inregion III in FIG. 1:

wild-type K65 (AAA) or polymorphic K65 (AAG), wild-type K66 (AAA) orpolymorphic K66 (AAG). wild-type D67 (GAC) or mutant N67 (AAC),wild-type T69 (ACT) or polymorphic T69 (ACA), mutant D69 (GAT) or N69(AAT) or A69 (GCT), wild-type K70 (AAA), polymorphic K70 (AAG) or mutantR70 (AGA).

Particularly preferred embodiments of the present invention include aset of probes for codons 74-75 comprising at least one, preferably atleast two, probes for targetting at least one, preferably at least two,nucleotide changes in any of the following codons as represented inregion IV of FIG. 1:

wild-type K73 (AAA) or polymorphic K73 (AAG), wild-type L74 (TTA) ormutant V74 (GTA), wild-type V75 (GTA) or polymorphic V75 (GTG) or mutantT75 (ACA), wild-type D76 (GAT) or polymorphic D76 (GAC).

Particularly preferred embodiments of the present invention include aset of probes for codon 151 comprising at least one, preferably at leasttwo, probes for targetting at least one, preferably at least two,nucleotide changes in any of the following codons as represented inregion V of FIG. 1:

wild-type L149 (CTT) or polymorphic L149 (CTC) or L149 (CTG), wild-typeP150 (CCA) or polymorphic P150 (CCG), wild-type Q151 (CAG) or mutantMI51 (ATG) or L151 (CTG) or polymorphic Q151 (CAA).

Particularly preferred embodiments of the present invention include aset of probes for codon 181-184 comprising at least one, preferably atleast two, probe(s) for targetting at least one, preferably at leasttwo, nucleotide changes in any of the following codons as represented inregion VI of FIG. 1:

wild-type Y181 (TAT) or mutant C181 (TGT), wild-type Q182 (CAA) orpolymorphic Q182 (CAG), wild-type Y183 (TAC) or polymorphic Y183 (TAT),wild-type M184 (ATG) or mutant V184 (GTG) or I184 (ATA) or G184 (AGG),wild-type D185 (GAT) or polymorphic D185 (GAC), wild-type D186 (GAT) orpolymorphic E186 (GAG), wild-type L187 (TTA) or polymorphic G187 (GGA)or V187 (GTA).

Particularly preferred embodiments of the present invention include aset of probes for codon 215 comprising at least one, preferably at leasttwo, probe(s) for targetting at least one, preferably at least two,nucleotide changes in any of the following codons as represented inregion VII of FIG. 1

wild-type G213 (GGA) or polymorphic G213 (GGG), wild-type F214 (TTT) orpolymorphic F214 (TTC) or L214 (CTT) or L214 (TTA), wild-type T215 (ACC)or polymorphic T215 (ACT), mutant Y215 (TAC) or F215 (TTC).

Particularly preferred embodiments of the present invention include aset of probes for codon 219 comprising at least one, preferably at leasttwo, probe(s) for targetting at least one, preferably at least two,nucleotide changes in any of the following codons as represented inregion VIII of FIG. 1:

wild-type D218 (GAC) or polymorphic D218 (GAT), wild-type K219 (AAA) orpolymorphic K219 (AAG) or mutant Q219 (CAA) or E219 (GAA), wild-typeK220 (AAA) or polymorphic K220 (AAG).

Examples of probes of the invention are represented in Tables 3 and 4,and FIGS. 2 and 3. In Table 3, the probes withheld after selection areindicated using the letter "y". These probes of the invention aredesigned for attaining optimal performance under the same hybridizationconditions so that they can be used in sets of at least 2 probes forsimultaneous hybridization; this highly increases the usefulness ofthese probes and results in a significant gain in time and labour.Evidently, when other hybridization conditions would be preferred, allprobes should be adapted accordingly by adding or deleting a number ofnucleotides at their extremities. It should be understood that theseconcommitant adaptations should give rise to essentially the sameresult, namely that the respective probes still hybridize specificallywith the defined target. Such adaptations might also be necessary if theamplified material should be RNA in nature and not DNA as in the casefor the NASBA (nucleic acid sequence-based amplification) system.

The selection of the preferred probes of the present invention is basedon a reverse hybridization assay using immobilized oligonucleotideprobes present at distinct locations on a solid support. Moreparticularly the selection of preferred probes of the present inventionis based on the use of the Line Probe Assay (LiPA) principle which is areverse hybridization assay using oligonucleotide probes immobilized asparallel lines on a solid support strip (Stuyver et al. 1993;international application WO 94/12670). This approach is particularlyadvantageous since it is fast and simple to perform. The reversehybridization format and more particularly the LiPA approach has manypractical advantages as compared to other DNA techniques orhybridization formats, especially when the use of a combination ofprobes is preferable or unavoidable to obtain the relevant informationsought.

It is to be understood, however, that any other type of hybridizationassay or format using any of the selected probes as described further inthe invention, is also covered by the present invention.

The reverse hybridization approach implies that the probes areimmobilized to certain locations on a solid support and that the targetDNA is labelled in order to enable the detection of the hybrids formed.

Methods for detecting nucleotide changes in RT genes of other viruseswhich have been found to harbour a pattern of drug-resistance mutationsimilar to the one observed for HIV based on the same principles as setout in the present invention should be understood as also being coveredby the scope of the present invention.

The following definitions serve to illustrate the terms and expressionsused in the present invention.

The term "antiviral drugs" refers particularly to an antiviralnucleoside analog or any other RT inhibitor. Examples of such antiviraldrugs and the mutation they may cause in the HIV-RT gene are disclosedin Schinazi et al., 1994 and Mellors et al., 1995. The contents of thelatter two documents particularly are to be considered as forming partof the present invention. The most important antiviral drugs focussed atin the present invention are disclosed in Tables 1 to 2.

The term "drug-induced mutation" refers to a mutation in the HIV RT genewhich provokes a reduced susceptibility of the isolate to the respectivedrug.

The target material in the samples to be analysed may either be DNA orRNA, e.g.: genomic DNA, messenger RNA, viral RNA or amplified versionsthereof. These molecules are also termed polynucleic acids.

It is possible to use genomic DNA or RNA molecules from HIV samples inthe methods according to the present invention.

Well-known extraction and purification procedures are available for theisolation of RNA or DNA from a sample (f.i. in Maniatis et al.,Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring HarbourLaboratory Press (1989)).

The term "probe" refers to single stranded sequence-specificoligonucleotides which have a sequence which is complementary to thetarget sequence to be detected.

The term "target sequence" as referred to in the present inventiondescribes the nucleotide sequence of the wildtype, polymorphic or druginduced variant sequence of the RT gene to be specifically detected by aprobe according to the present invention. This nucleotide sequence mayencompass one or several nucleotide changes. Target sequences may referto single nucleotide positions, codon positions, nucleotides encodingamino acids or to sequences spanning any of the foregoing nucleotidepositions. In the present invention said target sequence often includesone or two variable nucleotide positions. It is to be understood thatthe complement of said target sequence is also a suitable targetsequence in some cases. The target sequences as defined in the presentinvention provide sequences which should be complementary to the centralpart of the probe which is designed to hybridize specifically to saidtarget region.

The term "complementary" as used herein means that the sequence of thesingle stranded probe is exactly the (inverse) complement of thesequence of the single-stranded target, with the target being defined asthe sequence where the mutation to be detected is located.

Since the current application requires the detection of single basepairmismatches, very stringent conditions for hybridization are required,allowing in principle only hybridization of exactly complementarysequences. However, variations are possible in the length of the probes(see below), and it should be noted that, since the central part of theprobe is essential for its hybridization characteristics, possibledeviations of the probe sequence versus the target sequence may beallowable towards head and tail of the probe, when longer probesequences are used. These variations, which may be conceived from thecommon knowledge in the art, should however always be evaluatedexperimentally, in order to check if they result in equivalenthybridization characteristics than the exactly complementary probes.

Preferably, the probes of the invention are about 5 to 50 nucleotideslong, more preferably from about 10 to 25 nucleotides. Particularlypreferred lengths of probes include 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24 or 25 nucleotides. The nucleotides as used in thepresent invention may be ribonucleotides, deoxyribonucleotides andmodified nucleotides such as inosine or nucleotides containing modifiedgroups which do not essentially alter their hybridisationcharacteristics.

Probe sequences are represented throughout the specification as singlestranded DNA oligonucleotides from the 5' to the 3' end. It is obviousto the man skilled in the art that any of the below-specified probes canbe used as such, or in their complementary form, or in their RNA form(wherein T is replaced by U).

The probes according to the invention can be prepared by cloning ofrecombinant plasmids containing inserts including the correspondingnucleotide sequences, if need be by cleaving the latter out from thecloned plasmids upon using the adequate nucleases and recovering them,e.g. by fractionation according to molecular weight. The probesaccording to the present invention can also be synthesized chemically,for instance by the conventional phospho-triester method.

The term "solid support" can refer to any substrate to which anoligonucleotide probe can be coupled, provided that it retains itshybridization characteristics and provided that the background level ofhybridization remains low. Usually the solid substrate will be amicrotiter plate, a membrane (e.g. nylon or nitrocellulose) or amicrosphere (bead) or a chip. Prior to application to the membrane orfixation it may be convenient to modify the nucleic acid probe in orderto facilitate fixation or improve the hybridization efficiency. Suchmodifications may encompass homopolymer tailing, coupling with differentreactive groups such as aliphatic groups, NH₂ groups, SH groups,carboxylic groups, or coupling with biotin, haptens or proteins.

The term "labelled" refers to the use of labelled nucleic acids.Labelling may be carried out by the use of labelled nucleotidesincorporated during the polymerase step of the amplification such asillustrated by Saiki et al. (1988) or Bej et al. (1990) or labelledprimers, or by any other method known to the person skilled in the art.The nature of the label may be isotopic (³² P, ³⁵ S, etc.) ornon-isotopic (biotin, digoxigenin, etc.).

The term "primer" refers to a single stranded oligonucleotide sequencecapable of acting as a point of initiation for synthesis of a primerextension product which is complementary to the nucleic acid strand tobe copied. The length and the sequence of the primer must be such thatthey allow to prime the synthesis of the extension products. Preferablythe primer is about 5-50 nucleotides long. Specific length and sequencewill depend on the complexity of the required DNA or RNA targets, aswell as on the conditions of primer use such as temperature and ionicstrength.

The fact that amplification primers do not have to match exactly withthe corresponding template sequence to warrant proper amplification isamply documented in the literature (Kwok et al., 1990).

The amplification method used can be either polymerase chain reaction(PCR; Saiki et al., 1988), ligase chain reaction (LCR; Landgren et al.,1988; Wu & Wallace, 1989; Barany, 1991), nucleic acid sequence-basedamplification (NASBA; Guatelli et al., 1990; Compton, 1991),transcription-based amplification system (TAS; Kwoh et al., 1989),strand displacement amplification (SDA; Duck, 1990; Walker et al., 1992)or amplification by means of Qβ replicase (Lizardi et al., 1988; Lomeliet al., 1989) or any other suitable method to amplify nucleic acidmolecules known in the art.

The oligonucleotides used as primers or probes may also comprisenucleotide analogues such as phosphorothiates (Matsukura et al., 1987),alkylphosphorothiates (Miller et al., 1979) or peptide nucleic acids(Nielsen et al., 1991; Nielsen et al., 1993) or may containintercalating agents (Asseline et al., 1984).

As most other variations or modifications introduced into the originalDNA sequences of the invention these variations will necessitateadaptions with respect to the conditions under which the oligonucleotideshould be used to obtain the required specificity and sensitivity.However the eventual results of hybridisation will be essentially thesame as those obtained with the unmodified oligonucleotides.

The introduction of these modifications may be advantageous in order topositively influence characteristics such as hybridization kinetics,reversibility of the hybrid-formation, biological stability of theoligonucleotide molecules, etc.

The "sample" may be any biological material taken either directly fromthe infected human being (or animal), or after culturing (enrichment).Biological material may be e.g. expectorations of any kind,broncheolavages, blood, skin tissue. biopsies, sperm, lymphocyte bloodculture material, colonies, liquid cultures, faecal samples, urine etc.

The sets of probes of the present invention will include at least 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30 or more probes. Said probes may beapplied in two or more distinct and known positions on a solidsubstrate. Often it is preferable to apply two or more probes togetherin one and the same position of said solid support.

For designing probes with desired characteristics, the following usefulguidelines known to the person skilled in the art can be applied.

Because the extent and specificity of hybridization reactions such asthose described herein are affected by a number of factors, manipulationof one or more of those factors will determine the exact sensitivity andspecificity of a particular probe, whether perfectly complementary toits target or not. The importance and effect of various assayconditions, explained further herein, are known to those skilled in theart.

The stability of the [probe:target] nucleic acid hybrid should be chosento be compatible with the assay conditions. This may be accomplished byavoiding long AT-rich sequences, by terminating the hybrids with G:Cbase pairs, and by designing the probe with an appropriate Tm. Thebeginning and end points of the probe should be chosen so that thelength and % GC result in a Tm about 2-10° C. higher than thetemperature at which the final assay will be performed. The basecomposition of the probe is significant because G-C base pairs exhibitgreater thermal stability as compared to A-T base pairs due toadditional hydrogen bonding. Thus, hybridization involving complementarynucleic acids of higher G-C content will be stable at highertemperatures.

Conditions such as ionic strength and incubation temperature under whicha probe will be used should also be taken into account when designing aprobe. It is known that hybridization will increase as the ionicstrength of the reaction mixture increases, and that the thermalstability of the hybrids will increase with increasing ionic strength.On the other hand, chemical reagents, such as formamide, urea, DMSO andalcohols, which disrupt hydrogen bonds, will increase the stringency ofhybridization. Destabilization of the hydrogen bonds by such reagentscan greatly reduce the Tm. In general, optimal hybridization forsynthetic oligonucleotide probes of about 10-50 bases in length occursapproximately 5° C. below the melting temperature for a given duplex.Incubation at temperatures below the optimum may allow mismatched basesequences to hybridize and can therefore result in reduced specificity.

It is desirable to have probes which hybridize only under conditions ofhigh stringency. Under high stringency conditions only highlycomplementary nucleic acid hybrids will form; hybrids without asufficient degree of complementarity will not form. Accordingly, thestringency of the assay conditions determines the amount ofcomplementarity needed between two nucleic acid strands forming ahybrid. The degree of stringency is chosen such as to maximize thedifference in stability between the hybrid formed with the target andthe nontarget nucleic acid. In the present case, single base pairchanges need to be detected, which requires conditions of very highstringency.

The length of the target nucleic acid sequence and, accordingly, thelength of the probe sequence can also be important. In some cases, theremay be several sequences from a particular region, varying in locationand length, which will yield probes with the desired hybridizationcharacteristics. In other cases, one sequence may be significantlybetter than another which differs merely by a single base. While it ispossible for nucleic acids that are not perfectly complementary tohybridize, the longest stretch of perfectly complementary base sequencewill normally primarily determine hybrid stability. Whileoligonucleotide probes of different lengths and base composition may beused, preferred oligonucleotide probes of this invention are betweenabout 5 to 50 (more particularely 10-25) bases in length and have asufficient stretch in the sequence which is perfectly complementary tothe target nucleic acid sequence.

Regions in the target DNA or RNA which are known to form strong internalstructures inhibitory to hybridization are less preferred. Likewise,probes with extensive self-complementarity should be avoided. Asexplained above, hybridization is the association of two single strandsof complementary nucleic acids to form a hydrogen bonded double strand.It is implicit that if one of the two strands is wholly or partiallyinvolved in a hybrid that it will be less able to participate information of a new hybrid. There can be intramolecular andintermolecular hybrids formed within the molecules of one type of probeif there is sufficient self complementarity. Such structures can beavoided through careful probe design. By designing a probe so that asubstantial portion of the sequence of interest is single stranded, therate and extent of hybridization may be greatly increased. Computerprograms are available to search for this type of interaction. However,in certain instances, it may not be possible to avoid this type ofinteraction.

Standard hybridization and wash conditions are disclosed in theMaterials & Methods section of the Examples. Other conditions are forinstance 3× SSC (Sodium Saline Citrate), 20% deionized FA (Formamide) at50° C.

Other solutions (SSPE (Sodium saline phosphate EDTA), TMACl (Tetramethylammonium Chloride), etc.) and temperatures can also be used providedthat the specificity and sensitivity of the probes is maintained. Ifneed be, slight modifications of the probes in length or in sequencehave to be carried out to maintain the specificity and sensitivityrequired under the given circumstances.

In a more preferential embodiment, the above-mentioned polynucleic acidsfrom step (i) or (ii) are hybridized with at least two, three, four,five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteenfifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more of theabove-mentioned target region specific probes, preferably with 5 or 6probes, which, taken together, cover the "mutation region" of the RTgene.

The term "mutation region" means the region in the HIV RT gene sequencewhere most of the mutations responsible for antiviral drug resistance orother observed polymorphisms are located. A preferred part of thismutation region is represented in FIG. 1. This mutation region can bedivided into 8 important parts: drug induced variations andpolymorphisms located within aa positions 38 to 44 of RT gene, druginduced variations and polymorphisms located within aa positions 47 to53 of RT gene, drug induced variations and polymorphisms located withinaa positions 65 to 72 of the RT gene, drug induced variations andpolymorphisms located within aa positions 73 to 77 of the RT gene.drug-induced variations and polymorphisms located within aa positions148 to 154 of the RT gene, drug-induced variations and polymorphismslocated within aa positions 180 to 187 of the RT gene, drug inducedvariations and polymorphisms located within aa positions 212 to 216 ofthe RT gene and drug induced variations and polymorphisms located withinaa positions 217 to 220 of the RT gene.

Since some mutations may be more frequently occurring than others, e.g.in certain geographic areas or in specific circumstances (e.g. ratherclosed communities) it may be appropiate to screen only for specificmutations, using a selected set of probes as indicated above. This wouldresult in a more simple test, which would cover the needs under certaincircumstances.

In order to detect the antiviral drug RT resistance pattern with theselected set of oligonucleotide probes, any hybridization method knownin the art can be used (conventional dot-blot, Southern blot, sandwich,etc.).

However, in order to obtain fast and easy results if a multitude ofprobes are involved, a reverse hybridization format may be mostconvenient.

In a preferred embodiment the selected set of probes are immobilized toa solid support in known distinct locations (dots, lines or otherfigures). In another preferred embodiment the selected set of probes areimmobilized to a membrane strip in a line fashion. Said probes may beimmobilized individually or as mixtures to delineated locations on thesolid support.

A specific and very user-friendly embodiment of the above-mentionedpreferential method is the LiPA method, where the above-mentioned set ofprobes is immobilized in parallel lines on a membrane, as furtherdescribed in the examples.

The invention also provides for any probes and primer sets designed tospecifically detect or amplify specifically these RT gene polymorphisms,and any method or kits using said primer and probes sets.

The invention further provides for any of the probes as described above,as well as compositions comprising at least one of these probes.

The invention also provides for a set of primers allowing amplificationof the mutation region of the RT gene in general.

Primers may be labeled with a label of choice (e.g. biotine). Differentprimer-based target amplification systems may be used, and preferablyPCR-amplification, as set out in the examples. Single-round or nestedPCR may be used.

The invention also provides for a kit for inferring the nucleotidesequence at codons of interest in the HIV RT gene and/or the amino acidscorresponding to these codons and/or the antiviral drug resistancespectrum of HIV isolates present in a biological sample comprising thefollowing components:

(i) when appropiate, a means for releasing, isolating or concentratingthe polynucleic acids present in said sample;

(ii) when appropriate, at least one of the above-defined set of primers;

(iii) at least two of the probes as defined above, possibly fixed to asolid support;

(iv) a hybridization buffer, or components necessary for producing saidbuffer;

(v) a wash solution, or components necessary for producing saidsolution;

(vi) when appropriate, a means for detecting the hybrids resulting fromthe preceding hybridization.

(vii) when appropriate, a means for attaching said probe to a solidsupport.

The term "hybridization buffer" means a buffer enabling a hybridizationreaction to occur between the probes and the polynucleic acids presentin the sample, or the amplified products, under the appropiatestringency conditions.

The term "wash solution" means a solution enabling washing of thehybrids formed under the appropiate stringency conditions.

A line probe assay (LiPA) was designed for the screening for variationsat interesting amino acids in the HIV RT gene. The principle of theassay is based on reverse hybridization of an amplified polynucleic acidfragment such as a biotinylated PCR fragment of the HIV RT gene ontoshort oligonucleotides. The latter hybrid can then, via abiotine-streptavidine coupling, be detected with a non-radioactivecolour developing system.

The present invention further relates to a reverse hybridization methodwherein said oligonucleotide probes are immobilized, preferably on amembrane strip.

The present invention also relates to a composition comprising any ofthe probes as defined in Tables 3 and 4 or FIGS. 2 and 3.

The present invention relates also to a kit for inferring the HIV RTresistance spectrum of HIV in a biological sample, coupled to theidentification of the HIV isolate involved, comprising the followingcomponents:

(i) when appropiate, a means for releasing, isolating or concentratingthe polynucleic acids present in the sample;

(ii) when appropriate, at least one of the sets of primers as definedabove;

(iii) at least one of the probes as defined above, possibly fixed to asolid support;

(iv) a hybridization buffer, or components necessary for producing saidbuffer;

(v) a wash solution, or components necessary for producing saidsolution;

(vi) when appropriate, a means for detecting the hybrids resulting fromthe preceding hybridization;

(vii) when appropriate, a means for attaching said probe to a solidsupport

The following examples only serve to illustrate the present invention.These examples are in no way intended to limit the scope of the presentinvention.

Table 1: Mutations in HIV-1 RT gene associated with resistance againstnucleoside RT inhibitors. More details are given in Mellors et al.,1995.

Table 2: Mutations in HIV-1 RT gene associated with resistance againstHIV-1 specific RT inhibitors. For more details see Mellors et al., 1995.

Abbreviations in Table 1 and 2:

AZT: 3'-azido-2'3'-dideoxythymidine

ddC: 2'3'-dideoxycytidine

ddI: 2'3'-dideoxyinosine

3TC: 3'dideoxy-3'-thiacytidine

FTC: 2'3'-dideoxy-5-fluoro-3'-thiacytidine

L'697,593: 5-ethyl-6-methyl-3-(2-phthalimido-ethyl)pyridin-2(1H)-one

L'697,661: 3-Il(-4,7-dichloro-1,3-benzoxazol-2-yl)methylamino-5-ethyl-6-methylpyridin-2(1H)-one

Nevirapine:1l-cyclopropyl-5,1l-dihydro-4-methyl-6H-dipyridol(3,2-b:2',3'-e)diazepin-6-one

TIBO R82150:(+)-(5S)-4,5,6,7,-tetrahydro-5-methyl-6-(3-methyl-2butenyl)imidazo(4,5,1-)k)(1,4)-benzodiazepin-2(1H)-thione

TIBO 82913:(+)-(5S)-4,5,6,7,-tetrahydro-9-chloro-5-methyl-6-(3-methyl-2-butenyl)imidazo(4,5,1kj)-(1,4)benzo-diazepin-2(1H)-thione

TSAO-m² T:(2',5'-bis-o-(tert-buthyldimethylsilyl)-3'-spiro-5'-(4'-amino-1',2'-oxathiole-2',2'-dioxide)

U90152:1-(3-(1-methylethyl)-amino)-2-pyridinyl)-4-(5-(methylsulphonyl)-amino)-1H-indol-2yl)-carbonyl)-piperazine

Table 3: HIV RT wild-type and drug resistance mutation probes. Theprobes witheld after selection are indicated as "y".

Table 4: Prediction and prevalence of LiPA probe reactivity. Probe namescorresponding with the selected motifs are presented in the left column,with the relevant part of each probe shown under the consensus. Theprevalence of these motives, determined using panels of European and USsera, is given in the middle column.

The right column indicates the corresponding strips of FIGS. 2A-F andthe accession number of the reference panel clone used to obtain thisreactivity.

EXAMPLES Example 1

a. Materials and Methods.

Plasma samples were taken from HIV type-1 infected patients and storedat -20° C. until use. Patients were treated with AZT, ddI, ddC, D4T,3TC, or several combinations of these prodrugs. The European serumsamples tested were randomly selected. For the US serum collection, onlythe first sample from a follow-up series was taken. Some of these USpatients were treated, others were not treated.

HIV RNA was prepared using the guanidinium-phenol procedure. Fifty μlplasma was mixed with 150 μl Trizol®LS Reagent (Life Technologies, Gent,Belgium) at room temperature (volume ratio: 1 unit sample/3 unitsTrizol). Lysis and denaturation occured by carefully pipetting up anddown several times, followed by an incubation step at room temperaturefor at least 5 minutes. Fourty μl CHCl₃ was added and the mixture wasshaken vigorously by hand for at least 15 seconds, and incubated for 15minutes at room temperature. The samples were centrifuged at maximum12,000 g for 15 minutes at 4° C., and the colourless aquous phase wascollected and mixed with 100 μl isopropanol. To visualize the minuteamounts of viral RNA, 20 μl of 1 μg/μl Dextran T500 (Pharmacia) wasadded, mixed and left at room temperature for 10 minutes. Followingcentrifugation at max. 12,000 g for 10 minutes at 4° C. and aspirationof the supernatant, the RNA pellet was washed with 200 μl ethanol, mixedby vortexing and collected by centrifugation at 7,500 g for 5 minutes at4° C. Finally the RNA pellet was briefly air-dryed and stored at -20° C.

For cDNA synthesis and PCR amplification, the RNA pellet was dissolvedin 15 μl random primers (20 ng/μl, pdN₆, Pharmacia), prepared inDEPC-treated or HPLC grade water. After denaturation at 70° C. for 10minutes, 5 μl cDNA mix was added, composed of 4 μl 5×AMV-RT buffer (250mM Tris.HCl pH 8.5, 100 mM KCl, 30 mM MgCl₂, 25 mM DTT), 0.4 μL 25 mMdXTPs, 0.2 μl or 25 U Ribonuclease Inhibitor (HPRI, Amersham), and 0.3μl or 8 U AMV-RT (Stratagene). cDNA synthesis occured during the 90minutes incubation at 42° C. The HIV RT gene was than amplified usingthe following reaction mixture: 5 μl cDNA, 4.5 μl 10×Taq buffer, 0.3 μl25 mM dXTPs, 1 μl (10 pmol) of each PCR primer, 38 μl H₂ O, and 0.2 μl(1 U) Taq. The primers for amplification had the following sequence:outer sense RT-9: 5' bio-GTACAGTATTAGTAGGACCTACACCTGTC 3' (SEQ ID NO96); nested sense RT-1: 5' bio-CCAAAAGTTAAACAATGGCCATTGACAGA 3' (SEQ IDNO 97); nested antisense RT-4: 5' bio-AGTTCATAACCCATCCAAAG 3' (SEQ ID NO98); and outer antisense primer RT-12: 5'bio-ATCAGGATGGAGTTCATAACCCATCCA 3' (SEQ ID NO 99). Annealing occured at57° C., extension at 72° C. and denaturation at 94° C. Each step of thecycle took 1 minute, the outer PCR contained 40 cycles, the nested round35. Nested round PCR products were analysed on agarose gel and onlyclearly visible amplification products were used in the LiPA procedure.Quantification of viral RNA was obtained with the HIV Monitor™test(Roche, Brussels, Belgium).

Selected PCR products, amplified without 5' biotine primers, were clonedinto the pretreated EcoRV site of the pGEMT vector (Promega).Recombinant clones were selected after α-complementation and restrictionfragment length analysis, and sequenced with plasmid primers andinternal HIV RT primers. Other biotinylated fragments were directlysequenced with a dye-terminator protocol (Applied Biosystems) using theamplification primers. Alternatively, nested PCR was carried out withanalogs of the RT-4 and RT-1 primers, in which the biotine group wasreplaced with the T7- and SP6-primer sequence, respectively. Theseamplicons were than sequenced with an SP6- and T7-dye-primer procedure.Sequence information was submitted to the GENBANK.

Probes were designed to cover the different polymorphisms and druginduced mutations. In principle, only probes that discriminated betweenone single nucleotide variation were retained. However, for certainpolymorphisms at the extreme ends of the the probe, cross-reactivity wastolerated. Specificity was reached for each probe individually afterconsidering the % (G+C), the probe lenght, the final concentration ofthe buffer components, and hybridization temperature. Optimized probeswere provided enzymatically with a poly-T-tail using the TdT (Pharmacia)in a standard reaction condition, and purified via precipitation. Probepellets were disolved in standard saline citrate (SSC) buffer andapplied as horizontal parallel lines on a membrane strip. Control linesfor amplification (probe 5' CCACAGGGATGGAAAG 3', HIV RT aa 150 to aa155) and conjugate incubation (biotinylated DNA) were applied alongside.After fixation of the probes onto the membranes by baking, membraneswere sliced into 4 mm strips.

To perform LiPA tests, equal amounts (10 μl) of biotinylatedamplification products and denaturation mixture (0.4 N NaOH/0.1% SDS)were mixed, followed by an incubation at room temperature for 5 minutes.Following this denaturation step, 2 ml hybridization buffer (2×SSC, 0.1%SDS, 50 mM Tris pH7.5) was added together with a membrane strip andhybridization was carried out at 39° C. for 30 min. Then, thehybridization mixture was replaced by stringent washing buffer (samecomposition as hybridisation buffer), and stringent washing occuredfirst at room temperature for 5 minutes and than at 39° C. for another25 minutes. Buffers were than replaced to be suitable for thestreptavidine alkaline phosphatase conjugate incubations. After 30minutes incubation at room temperature, conjugate was rinsed away andreplaced by the substrate components for alkaline phosphatase,Nitro-Blue-Tetrazolium and 5-Bromo-4-Chloro-3-Indolyl Phosphate. After30 minutes incubation at room temperature, probes where hybridizationoccured became visible because of the purple brown precipitate at thesepositions.

b. Results.

b.1 The HIV-1 RT Gene PCR and Selection of a Reference Panel.

PCR primers were chosen outside the target regions for probe design. Theamplified region located inside the nested primers covered the HIV-1 RTgene from codon 29 to codon 220. The primer design was based onpublished sequences from the HIV-1 genotype B clade. European and UnitedStates HIV-1 positive serum samples, stored appropriately (at -20° C.)without repeating freezing-thawing cycles, were PCR positive in 96% ofthe cases (not shown). The annealing temperature for the selectedprimers seemed to be crucial (57° C.). At 55° C., a second a specificamplicon of approximately 1500-bp was generated; and at 59° C. theamount of specific fragment decreased drastically. With the currentprimer combination, the corresponding RT region could be amplified fromisolates of the genotype A, C, D and F clade, but with a reducedsensitivity.

A total of 25 selected PCR fragments with the target polymorphisms andmutations were retained as reference panel and sequenced on bothstrands. The selection occurred during the evaluation of the probes, andthese samples originated from naive or drug-treated European or USpatients. Biotinylated PCR products from this panel (Accession NumberL78133 to L78157) were used to test probes for specificity andsensitivity.

b.2 Nucleotide Target Region for Probe Design and Probe Selection.

Table 4 and parts of FIG. 1 are a compilation of the natural anddrug-selected variability in the vicinity of aa 41, 69-70, 74-75, 184,215, and 219 of the HIV RT gene. To create this table and parts of thisfigure, the "National Centre for Biotechnology Information" database wassearched and all HIV-1 genome entries were retrieved and analyzed one byone. Only those entries displaying non-ambiguous sequence information inthe vicinity of the above-mentioned codons were retained for furtherinterpretation. It should be noted that the indicated variations do notimply that they occur in the same sequence: for example the variabilityobserved at codon 40 and 43 may occasionally occur together, but mostoften, if they occur, only one of them is found. In these 6 regions, atotal of 19 different third-letter and two first-letter (codon 43 AAGversus GAG and codon 214 TTT versus CTT) polymorphisms need to beincluded in the selection of wild type probes. Another 13 first-letterand/or second-letter variations are drug-induced and are the maintargets for the selection of probes (FIG. 1).

For the design of relevant probes, only those database motifs thatsystematically returned (highly prevalent motif) were included, whilescattered mutations which were found randomly (low prevalent motif, notshown) were ignored. Based on database sequences, seven motifs for codon41 (91.6% of all entrees), 6 for codon 69-70 (86.2%), 2 for codon 74-75(90.4%), 5 for codon 184 (96.6%), 9 for codon 215 (94.1%), and 2 forcodon 219 (88.2%) were selected (Table 4).

Probe names corresponding with the selected motifs are presented in theleft column of Table 4, with the relevant sequence part of each probeshown under the consensus. The prevalence of these motives was thandetermined using panels of European and US sera (Table 4).

In many cases, the database entries were not representative for thesamples tested. Upon analyzing the European and US samples, many werenot reactive with these database-selected probes. Upon sequencinganalysis of several of these unreactive PCR products, another 8 motifsbecame apparent, for which the corresponding probes were designed(41w20, 41m12, 70m13, 74w9, 74m6, 74m12, 184w24, 215m49). By includingthese newly designed motifs, negative results were markedly decreased;for all codon positions except codon 219, the total percentage ofreactivity exceeds 90%.

Another 4 probes were designed (41m11, 215m50, 219m7, and 219m9) becausetheir sequence motif was found in the cloned reference panel, althoughno reactivity with the tested plasma virus samples was found so far. Theexistence of these rare sequence motifs is explained by assuming thatthey exist at an extremely low frequency in the viral quasispecies,remaining undetectable by direct detection methods, but becomingapparent after cloning.

The sequence motif of probe 215m13 was generated in recombinant clonesby site-directed mutagenesis (not shown). The rational behind this probedesign was to determine whether the sequence combination of codon Y215(TAC) can occur in combination with L214 (CTT) in vivo. However, thislatter motif was not found in the plasma samples tested.

Four probes (41w15, 70w8, 215w29, 215w27) are in fact redundant, becausethey detect identical sequence motifs covered by other probes. However,the location of these redundant probes is slightly different to theirsequence-identical counterpart. These probes have the potential to avoidnegative results which might otherwise appear as a consequence of randommutations in the probe target area and can therfore increase thespecificity of recognition.

b.3 Probe Specificity and Sensitivity.

The 48 selected probes were applied separately on LiPA strips.Biotinylated PCR fragments generated from the reference panel ordirectly from plasma virus were alkali-denatured, the hybridizationbuffer and LiPA strips were added, and submitted to stringenthybridization and washing conditions. Positions where hybridizationoccurred were revealed by the biotine-streptavidine calorimetricdetection system. FIG. 2(A to F) shows the reactivity of these 48designed probes. In the right columns of Table 4, there is theindication of the corresponding strip in FIG. 2, and the accessionnumber of the reference panel clone used to obtain this reactivity. Thereactivities of these probes were concordant with the nucleotidesequences. False positive reactivities were observed only for probe41w19 (FIG. 2A.9) and for 70m3 (FIG. 2B.8), with extremely rare sequencemotifs 41m12 (prevalence less than 0.3%) and 70m16 (not experimentallyfound), respectively. Weak cross-reactivity, as was observed on probe41m13 with a 41m27 motif (FIG. 2A.10) was, in general, not tolerated inthe probe design. When occurring, however, it never influenced thegenotypic resistance interpretation.

b.4 Applicability of the LiPA in Patient Management.

We selected follow-up samples from three patients and analyzed the viralgenotype on the 48 LiPA probes. FIG. 3 illustrates the applicability ofgenotypic resistance measurement in conjunction with the analysis ofviral load and CD4 cell count. All three patients had a wild type virus(i.e. M41-T69-K70-L74-V75-M184-F214-T219-K219) strain in the samplecollected before anti-retroviral treatment. Codon positions that changedupon treatment are presented in FIG. 3.

From Patient 91007, 11 serum samples were analyzed, the first samplebeing collected 2 weeks before the start of therapy. The LiPA revealedthat before treatment, in a T215 context, two variants at codon position213 were predominantly present (GGG and GGA respectively detected byprobe 215w11 and 215w9/215w29). From week 50 until week 81, a mixture ofT215 and Y215 could be detected. Both variants at codon 213 were alsorepresented in the selected resistant genotypes (probes 215m17 and215m14 are positive). From week 94 onwards, only Y215 mutant virus couldbe detected. A nearly identical geno-conversion at codon 41 wasobserved, with the detection of mixtures (M41 and L41) from week 81until week 111; from week 126 onwards, only L41 could be found (stripsnot shown). CD4 values were highly variable. Nevertheless, a continuousdecrease in CD4 is apparent (p=0.019, linear regression analysis). Viralload also decreased initially. However, the direct response to thetreatment might have been missed in this follow-up series, since thefirst sample after the start of the treatment is at 32 weeks. From thanon, viral load increased.

Patient 94013 was treated with 3TC monotherapy from week 2 onwards. Atweek 10, a mixture of M184 and V184 could be detected. From week 14 on,only V184 was present. CD4 counts increased nearly 2.5-fold, with thehighest level at week 10. Viral load decreased spectacularly by 3 logunits. But from week 10 onwards, a slight but steady increase to week 23was noted. The decrease in CD4 and increase in viral load coincided withthe appearance of the V184 motif.

Patient 92021 was followed for 55 weeks. AZT treatment started at week10, followed by a supplemental ddC treatment from week 20 onwards. Thefirst sample was found to be reactive with probe 215w9/w29(F214T215=TTTACC), but trace amounts of reactivity with 215w53(L214T215=TTAACC) could be detected as well, indicating the presence ofat least two variants at that time. From week 19 onwards, the codonL214=TTA motif became more important. At week 42, the first sign ofgenotypic resistance could be detected by the presence of a F214Y215motif (TTTTAT). Finally at week 55, only F214Y215 could be detected. TheL214=TTA motif disappeared completely. At week 42, a mixture (K and R)at codon 70 was present, but at week 55, only R70 could be detected. Atweek 55, a mixture of codon 219 motifs (K and E) was found (strips notshown). CD4 initially increased, with a maximal effect during AZTmonotherapy peaking at week 21. From then on, a continuous decrease wasobserved. However, ten weeks of AZT treament did not result in a drop inviral load, since the values of week 16 and 19 were nearly unchanged. Itis only after start of the combination therapy (week 20) that the viralload dropped by 1.67 log. From this patient, it is tempting to assumethat L214T215 confers genotypic resistance to AZT treatment, and thatthe addition of ddC is necessary to induce the natural F214Y215genotype. The rise in CD4 cell count may be the consequence of the drugitself, and not from drug-induced protection (Levy et al. 1996).

c. Discussion

By adapting the previously designed LiPA technology (Stuyver et al.1993) for the HIV RT gene, the described assay format permits the rapidand simultaneous detection of wild type and drug-selected variantsassociated with the genotypic resistance for AZT, ddI, ddC, d4T, FTC and3TC. The Inno LiPA HIV drug-resistance strip provides information aboutthe genetic constitution of the RT gene in the vicinity of codon 41, 69,70, 74, 75, 184, 215, and 219 at the nucleotide and, hence, also at thededuced protein level. Essentially, the biotinylated RT PCR product ishybridized against immobilized specific oligonucleotides (Table 4),which are directed against the indicated codon variabilities. Followingthis reverse-hybridization, the oligonucleotide-biotinylated-PCR-strandhybrid is recognized by the streptavidine-alkaline phosphate conjugate,which then in turn converts the alkaline phosphate substrate into apurple brown precipitate.

Using this assay, we studied the specificity and reactivity of 48probes, covering 6 different regions. This combination should allow thereliable detection of most of the genetic resistance-related codoncombinations observed to date. Occasionally occurring mutations in thevicinity of the target codons, not taken into consideration during probedesign, may eventually prevent hybridization of the probes for aparticular target region. This problem is partially solved by theredundancy of probes at the most important codons. Results obtainedusing 358 HIV infected plasma samples showed that, depending on thecodon position under investigation, between 82.4% and 100% of thecombinations could be detected, or an average of 92.7%. It is importantto mention here that the assay was developed for resistence detection ofthe HIV-1 genotype B, and only limited information is currentlyavailable about the outcome of this assay with other genotypes. Sincethe amplification primer combination is more or less universal for allthe HIV-1 isolates, some of the indeterminate results may well be due tothe presence of non-genotype B virus strains.

So far, several assays for the detection of the wild-type anddrug-selected mutations in the HIV RT gene have been described. Theseinclude Southern blotting (Richman et al., 1991), primer-specific PCR(Larder et al., 1991), PCR-LDR (Frenkel et al., 1995), RNAse A mismatchcleaving (Galandez-Lopez et al., 1991), and hybridization againstenzyme-labeled probes (Eastman et al., 1995). The general advantage ofthe LiPA and other genotypic assays is the speed by which results areobtained when compared to phenotypic assays. The particular advantage ofour test is its multi-parameter (in this particular case multi-codon)format. Moreover, the assay can easily be extended not only for thescreening of the other RT-codons, but also for proteinase codonsassociated with resistance (Mellors et al., 1995). As was illustrated inFIG. 3, mixtures of wild-type and drug-selected mutations can bedetected easily. The detection limit for these mixtures is dependent onthe sensitivity of the probes, but reliable results can be obtained assoon as 5 to 10% of the minor component is present (not shown). We wereunable to provide reliable evidence for mixtures with any sequencingprotocol at the same sensitivity level.

Due to the large amount of variables that need to be included in theselection of specific probes (temperature of hybridization, ionicstrength of hybridization buffer, length of the probe, G+C content,strand polarity), it might occasionally occur that some of the probeswill show weak false positive reaction with related but hithertounreported sequences. In our experience, and if this occurred, this hasnever influenced the interpretation at the deduced aa level. In thecurrent selection of probes, all except two (41w19 and 70m3) wereretained on the basis of 100% specificity: as soon as one nucleotidediffers in the probe area, hybridization is abolished. Furtherfine-tuning of these two probes will therefore be necessary to obtainthe required specificity.

Accompanying polymorphisms in the vicinity of the target codons arefound with a rather high prevalence in wild-type virus strains, but notin mutant sequences. A partial list of such combinations is herebypresented: codon V74=GTA without polymorphism at codon 73, 75 or 76;codon V184=GTG without codon Q182=CAG; and codon F215=TTC withoutF214=TTC/TTA or L214=CTT. The most intriguing example is the following:L214T215 (CTTACC) is predicted for approximately 7.8% of the wild typesequences. The corresponding motif L214Y215 (CTTTAT) apparently does notexist in plasma virus. From the example shown in FIG. 3, it is clearthat selection of mutants is a very flexible and complex phenomena. Inthis particular case, viruses having codon F214 were replaced by a L214viral population in the AZT monotherapy period, but upon selecting forgenotypic drug resistance at codon 215, the original F214 configurationwas restored. Clearly, the selection for the Y215 genotype prohibits thepresence of a L214 genotype. Since no evidence has yet emerged that L214confers resistance to anti-retroviral compounds, the appearance of thisspecial mutant during the AZT monotherapy period is difficult tointerpret. More research will certainly be necessary to clarify thisissue. But if L214 should indeed provide low-level genotypic resistanceto AZT treatment, approximately 7.8% of the naive infections will notbenefit from initial AZT monotherapy.

Since antiviral treatment can result in a marked extension of lifeexpectancy for HIV infected patients, it is of utmost importance to findthe best drug regimen for each individual separately. Therefore,monitoring of the magnitude and duration of the virus load and CD4 cellchanges is a prerequisite. However, knowledge concerning the geneticconstitution of the virus may also be an important factor in designingoptimal treatment schedules. Optimizing therapies making good use ofavailable information (viral load, CD4 cell count, genetic resistance)has remained largely unexploited. If this was partially due to thecomplexity of screening for all the mutational events, theabove-described LiPA technology should remove one key obstacle.

In conclusion, we have described a genotypic assay for the detection ofwild type and drug selected codons in the HIV RT gene. The combinationof the assay result along with viral load and CD4 cell monitoring shouldpermit better design of patient-dependent optimal treatment schedules.

Example 2

Multi-Drug Resistant (MDR) HIV-1 isolates have been described. These MDRisolates are characterized by having mutations in their genome, comparedto the wild type HIV-1 genome, which result in a set of amino acidchanges. A key mutation leading to multi-drug resistance was found to belocalized in codon 151 of the HIV-1 RT gene. Consequently, and asdetecting these MDR isolates is clinically important, we designed probesrecognizing wild-type (probe 151w2) and mutant (probes 151m4 and 151m19)HIV-1 isolates. Furthermore, the presence of polymorphisms in the directvicinity of codon 151 (codon 149) and at codon 151 have been described.Therefore, we also designed two additional probes (probes 151w6 and151w11) which detect these polymorphisms (FIG. 4 and Table 3).

Treatment with non-nucleoside analogues, such as Nevirapine (BoehringerIngelheim), selects for several amino acid changes in conserved regionsof the HIV-1 RT gene. One of the most important amino acid changes isY181C, a codon change that confers high level resistance. As thedetection of this mutation is also clinically important, we designedprobes recognizing the wild-type (181w3 and 181w5) and mutant (181m7)isolates (FIG. 4 and Table 3).

FIG. 4 shows the application of the selected probes for codon 151 and181. The position of the probes on the strips is indicated on the rightside of the strips. LiPA strips were incubated with sequence-confirmedPCR fragments, extracted and amplified from: a wild type HIV-1 isolate(strip 1), a wild type HIV-1 isolate with a polymorphism at codon 151(strip 2) or codon 149 (strip 3), a multi-drug resistant HIV-1 isolate(strip 4) with no information about codon 181 and a non-nucleosideanalogue-treated HIV-1 isolate which remained wild type at codon151(strip 5).

                  TABLE 1                                                         ______________________________________                                        AZT          M41L       ATG to TTG or CTG                                                               D67N GAC to AAC                                        K70R AAA to AGA                                                               T215Y ACC to TAC                                                              T215F ACC to TTC                                                              K219Q AAA to CAA                                                              K219E AAA to GAA                                                             ddl K65R AAA to AGA                                                            L74V TTA to GTA                                                               V75T GTA to ACA                                                               M184V ATG to GTG                                                             ddC K65R AAA to AGA                                                            T69D ACT to GAT                                                               L74V TTA to GTA                                                               V75T GTA to ACA                                                               M184V ATG to GTG                                                              Y215C TTC to TGC                                                             d4T I50T ATT to ACT                                                            V75T GTA to ACA                                                              3TC or M184V ATG to GTG or GTA                                                FTC M184I ATG to ATA                                                          1592U89 K65R AAA to AGA                                                        L74V TTA to GTA                                                               Y115F TAT to TTT                                                              M184V ATG to GTG                                                           ______________________________________                                    

                  TABLE 2                                                         ______________________________________                                        Nevirapine      A98G       GCA to GGA                                            L100I TTA to ATA                                                              K103N AAA to AAC                                                              V106A GTA to GCA                                                              V108I GTA to ATA                                                              Y181C TAT to TGT                                                              Y181I TGT to ATT                                                              Y188C TAT to TGT                                                              G190A GGA to GCA                                                             TIBO L199I TTA to ATA                                                         R82150                                                                        TIBO L100I TTA to ATA                                                         R82913 K103N AAA to AAC                                                        V106A GTA to GCA                                                              E138K GAG to AAG                                                              Y181C TAT to TGT                                                              Y188H TAT to CAT                                                              Y188L TAT to TTA                                                             L697,593 K103N AAA to AAC                                                      Y181C TAT to TGT                                                             L697,661 A98G GCA to GGA                                                       L100I TTA to ATA                                                             L697,661 K101E AAA to GAA                                                      K103N AAA to AAC                                                              K103Q AAA to CAA                                                              V108I GTA to GCA                                                              V179D GTT to GAT                                                              V179E GTT to GAG                                                              Y181C TAT to TGT                                                             BHAP                                                                          U-90152 P236L CCT to CTT                                                      BHAP K101E AAA to GAA                                                         U-87201 K103N AAA to AAC                                                       Y181C TAT to TGT                                                              Y188H TAT to CAT                                                              E233V GAA to GTA                                                              P236L CCT to CTT                                                              K238T AAA to ACA                                                             BHAP L100I TTA to ATA                                                         U-88204 V106A GTA to GCA                                                       Y181C TAT to TGT                                                              Y181I TGT to ATT                                                             HEPT Y188C TAT to TGT                                                         E-EBU Y181C TAT to TGT                                                        E-EBU-dM Y106A GTA to GCA                                                     E-EPU and Y181C TAT to TGT                                                    E-EPSeU Y188C TAT to TGT                                                      a-APA Y181C TAT to TGT                                                        R18893                                                                        S-2720 G190E GGA to GAA                                                       TSAO E138K GAG to AAG                                                         BM + 51.0836 Y181C TAT to TGT                                               ______________________________________                                    

                                      TABLE 3                                     __________________________________________________________________________    HIV RT wild-type and drug resistance                                          Formula probe                                                                             Sequentie oligo                                                                              SEQ ID NO PROBE selection                          __________________________________________________________________________    wild-type probes for position M41                                             E40M41K43                                                                             41w7                                                                              AGAAATGGAAAAGGA                                                                              1 y                                                  E40M41K43 41w15 TGTACAGAAATGGAA 2 y                                           M41K43 41w16 AAATGGAAAAGGAAG 3                                                E40M41 41w18 TACAGAGATGGAAA 4                                                 E40M41K43 41w19 GTACAGAGATGGAAA 5                                             E40M41K43 41w20 AGAGATGGAAAAAGA 6 y                                           E40M41K43 41w30 AGAAATGGAGAAGGA 7 y                                           E40M41 41w31 ACAGAGATGGAAAA 8                                                 E40M41 41w32 GTACAGAGATGGAA 9 y                                               E40M41K43 41w33 CAGAGATGGAAAAG 10                                             E40M41K43 41w34 AGAAATGGAAAAAGA 11                                            E40M41K43 41w35 GAAATGGAAAAAGA 12                                             E40M41K43 41w36 CAGAAATGGAAAAAGA 13 y                                         E40M41K43 41w37 AGAAATGGAAAAAGAA 14                                         drug-induced variant probes for position L41                                  E40L41K43                                                                             41m8                                                                              AGAATTGGAAAAGGA                                                                              15                                                   E40L41K43 41m11 AGAGTTGGAAAAGGA 16 y                                          E40L41K43 41m12 AGAGCTGGAAAAGG 17 y                                           E40L41K43 41m13 AGAACTGGAAAAGG 18 y                                           E40L41K43 41m14 GAGCTGGAAAAGG 19                                              E40L41K43 41m21 ACAGAATTGGAAAAG 20 y                                          E40L41 41m22 ACAGAATTGGAAAA 21                                                E40L41 41m23 ACAGAACTGGAAAA 22                                                E40L41K43  41m24 AGAATTGGAAGAGG 23 y                                          E40L41E43  41m25 CAGAATTGGAAGAGG 24                                           E40L41E43 41m26 AGAATTGGAAGAGGA 25                                            E40L41E43 41m27 AGAACTGGAAGAGG 26 y                                           E40L41E43 41m28 CAGAACTGGAAGAGG 27                                            E40L41E43 41m29 AGAACTGGAAGAGGA 28                                          wild-type probes for positions I50 or V50 or T50                              K49I50  50w4                                                                              CAAAAATTGGGCCT 29 y                                                 R49I50 50w9 ATTTCAAGAATTGGG 30 y                                              K49V50 50w5 TTCAAAAGTTGGGC 31 y                                               K49I50 50w13 CAAAAATCGGGCCTG 32 y                                             K49T50 50w14 AAAAATCGGGCCTGA 33 y                                           wild-type probe for position D67                                              K64K65K66D67                                                                          67w4                                                                              AAAGAAGAAAGACAG                                                                              34 y                                               drug-induced variant probe for position N67                                   K64K65K66N67                                                                          67m19                                                                             ATAAAGAAAAAGAACAGTA                                                                          35 y                                               wild-type probes for positions T69 or K70                                     T69K70  70w1                                                                              AGTACTAAATGGAGAA                                                                             36 y                                                 D69K70 70w2 AGTGATAAATGGAGAA 37 y                                             T69K70 70w8 ACAGTACTAAATGGAG 38 y                                             K70K73 70w11 TAAATGGAGAAAAITAG 40                                           drug-induced variant probes for positions D69 or N69 or A69 or R70            D69R70  70m3                                                                              GTGATAGATGGAGAA                                                                              41                                                   T69R70 70m6 GTACTAGATGGAGA 42                                                 T69R70 70m12 AGTACTAGATGGAGA 43 y                                             T69R70 70m13 AGTACAAGATGGAGA 44 y                                             N69R70 70m14 CAGTAATAGATGGAG 45 y                                             A69R70 70m15 ACAGTGCTAGATGGA 46                                               A69R70 70m16 CAGTGCTAGATGGA 47 y                                              A69R70 70m17 CAGTGCTAGATGGA 48                                                D69R70 70m18 CAGTGATAGATGGA 49 y                                              D69R70 70m19 CAGTGATAGATGGAG 50                                               D69R70 70m20 AGTGATAGATGGAG 51                                                D69R70 70m21 AGTGATAGATGGAGA 52                                             wild-type probes for positions L74 or V75                                     K73L74V75D76                                                                          74w5                                                                              GAGAAAATTAGTAGATTT                                                                           53 y                                                 K73L74V75D76 74w8 AAAATTAGTAGACTTC 54 y                                       K73L74V75D76 74w9 GAGAAAGTTAGTGGATT 55                                      drug-induced variant probes for positions V74 or T75                          K73V74V75D76                                                                          74m6                                                                              AGAAAAGTAGTAGATTT                                                                            56 y                                                 K73L74T75D76 74m10 AAAATTAACAGATTTC 57                                        K73L74T75D76 74m11 GAAAATTAACAGATTT 58                                        K73L74T75D76 74m12 GAAAATTAACAGATTTC 59 y                                   wild-type probes for position Q151                                            P150Q151G152                                                                          151w2                                                                             CTTCCACAGGGATGG                                                                              60 y                                                 P150Q151G152 151w6 CTTCCACAAGGATGG 61 y                                       P150Q151G152 151w11 TGCTCCCACAGGGATG 62 y                                   drug-induced variant probe for position M151                                  P150M151G152                                                                          151m4                                                                             CTTCCAATGGGATGG                                                                              63 y                                                 P150M151G152 151m19 GCTTCCAATGGGATGG 64 y                                   wild-type probe for position Y181                                             Y181    181w3                                                                             AGTTATCTATCAATACAG                                                                           65 y                                               drug-induced variant probe for position C181                                  C181    181m7                                                                             AGTTATCTGTCAATAC                                                                             66 y                                               wild-type probes for position M184                                            Q182M184                                                                              184w11                                                                            TCAATACATGGATGAGG                                                                            67 y                                                 Q182M184 184w17 TCAGTACATGGATGAGG 68 y                                        Q182M184 184w18 ATCAATACATGGATGA 69                                           Q182M184 184w19 TCAGTACATGGATG 70                                             Q182M184 184w21 ATCAATATATGGATG 71 y                                          Q182M184 184w22 ATCAATATATGGATGA 72                                           Q182M184 184w23 TCAATATATGGATGA 73                                            Q182M184 184w24 TCAATACATGGACGA 74 y                                          Q182M184 184w25 CAATACATGGACGAT 75                                            Q182M184 184w26 TCAATACATGGACGAT 76                                         drug-induced variant probes for position V184 or I184                         Q182V184                                                                              184m12                                                                            CAATACGTGGATGAGGG                                                                            77 y                                                 I184 184m13 AATACATAGATGAT 78                                                 Q182I184 184m14 CAATACATAGATGAT 79                                            Q182I184 184m15 CAATACATAGATGATT 80                                           Q182V184 184m16 CAATACGTAGATGAT 81                                            Q182V184 184m20 TCAATACGTGGATGA 82                                            Q182I184 184m27 TCAATACATAGATGAT 83                                           Q182I184 184m28 ATCAATACATAGATGAT 84 y                                      wild-type probes for position T215                                            G213F214T215                                                                          215w9                                                                             GGATTTACCACACCA                                                                              85 y                                                 L214T215 215w10 GACTTACCACACCA 86 y                                           F214T215 215w11 GGTTTACCACACCA 87 y                                           F214T215 215w16 GATTTACCACACCA 88                                             T215 215w22 TTACTACACCAGAC 89 y                                               T215 215w24 TTACCACACCAGA 90                                                  G213L214T215 215w27 TGGGGACTTACCAC 91 y                                       G213F214T215 215w29 TGGGGATTTACCAC 92 y                                       G213F214T215 215w32 GGGGTTCACCACAC 93                                         G213F214T215 215w33 GGGATTCACCACAC 94 y                                       G213F214T215 215w34 GGGATTTACCACACCAG 95                                      G213L214T215 215w35 TGGGGACTTACCACACC 96                                      G213F214T215 215w36 TGGGGGTTTACCACACC 97                                      G213F214T215 215w37 GGGATTTACTACACCAG 98                                      G213L214T215 215w52 GGGATTAACCACAC 99                                         G213L214T215 215w53 GGGGATTAACCACA 100 y                                      G213L214T215 215w54 TGGGGATTAACCACA 101                                       G213L214T215 215w55 GGGGGTTAACCACA 102                                        G213L214T215 215w56 GGGGTTAACCACAC 103                                        G213L214T215 215w57 TGGGGGTTAACCAC 104                                        G213L214T215 215w65 GGGATTGACCACAC 105                                        G213L214T215 215w66 GGATTGACCACACC 106                                        G213L214T215 215w67 GGGATTGACCACA 107 y                                       G213L214T215 215w68 GGGACTGACCACA 108 y                                       G213L214T215 215w69 GGGACTGACCACAC 109                                        G213L214T215 215w70 TGGGGGTTAACCACA 110                                       G213L214T215 215w71 TGTGGTTAACCCCCA 111 y                                     G213L214T215 215w51 GGGGCTTACCACAC 112                                      drug-induced variant probes for position Y215 or F215                         G213L214Y215                                                                          215m13                                                                            GGACTTTACACACC 113 y                                                G213F214Y215 215m14 GGGTTTTACACACC 114 y                                      G213F214F215 215m15 GGATTTTTCACACCA 115                                       G213F214Y215 215m17 GGATTTTACACACC 116 y                                      G213F214Y215 215m38 GGGATTTTACACACCAG 117                                     G213F214F215 215m39 GGGATTTTTCACACCAG 118                                     G213F214Y215 215m40 GGGATTTTACACAC 119                                        G213F214Y215 215m41 GGGGATTTTACACA 120                                        G213F214Y215 215m43 CCCTAAAATGTGTG 121                                        G213F214F215 215m44 GGATTTTTCACACC 122                                        F214F215 215m45 GATTTTTCACACCA 123 y                                          G213F214F215 215m46 GGGATTTTTCACAC 124                                        G213F214Y215 215m42 CCCCTAAAATGTGT 125                                        F214Y215 215m47 GGTTTTATACACCA 126                                            G213F214Y215 215m48 GGGTTTTATACACC 127                                        G213F214Y215 215m49 GGGGTTTTATACAC 128 y                                      G213L214T215 215m50 GGGGGCTTACCACA 129 y                                      G213F214Y215 215m61 GGATTCTACACACC 130 y                                      F214Y215 215m62 GATTCTACACACC 131                                             G213F214Y215 215m63 GGATTCTACACAC 132                                         G213F214Y215 215m64 GGGATTCTACACAC 133                                        G213F214Y215 215m72 GGGTTTTATACCCC 134                                        F214Y215 215m73 GGTTTTATACCCC 135                                             F214Y215 215m74 GTTTTATACCCCA 136                                           wild-type probes for position K219                                            K219    219w1                                                                             ACCAGACAAAAAACA                                                                              137                                                  K219 219w2 ACCAGACAAAAAAC 138 y                                               K219 219w3 CACCAGACAAAAAAC 139                                                K219 219w13 CAGACAAGAAACAT 140                                                K219 219w14 CCAGACAAGAAACA 141                                                K219 219w15 ACCAGACAAGAAACA 142                                               K219 219w16 AGACAAAAAGCATC 143 y                                              K219 219w17 CAGACAAAAAGCAT 144                                                K219 219w18 CAGACAAAAAGCATC 145                                               K219 219w19 CCAGATAAAAAACA 146                                                K219 219w20 ACCAGATAAAAAAC 147                                                K219 219w21 CCCAGATAAAAAACA 148                                               K219 219w22 CCAGATAAAAAACATC 149                                              K219 219w23 CACCAGATAAAAAAC 150                                               K219 219w24 CAGACAAGAAACATC 151                                               K219 219w25 ACCAGACAAGAAAC 152                                              drug-induced variant probes for position Q219 or E219                         Q219    219m4                                                                             ACCAGACCAAAAACA                                                                              153                                                  E219 219m5 ACCAGACGAAAAACA 154                                                Q219 219m6 ACCAGATCAAAAACA 155                                                Q219 219m7 ACCAGATCAAAAAC 156 y                                               Q219 219m8 CACCAGATCAAAAAC 157                                                E219 219m9 ACCAGACGAAAAAC 158 y                                               E219 219m10 CCAGACGAAAAACA 159                                                Q219 219m11 CCAGACCAAAAACA 160                                                Q219 219m12 ACCAGACCAAAAAC 161                                              __________________________________________________________________________

                                      TABLE 4                                     __________________________________________________________________________    Prediction and prevalance of LiPA probe reactivity                                  concensus             prevalance               Corresponding            nucleic acid        amino acid                                                                            database  Europe                                                                             US   Rp   Figure                   __________________________________________________________________________    probe Codon 38-43           n=191/m=25                                                                              n=306                                                                              n=52 n=25 strip                                                                              Acc.Nb              __________________________________________________________________________          TGTACAGAAATGGAAAAG                                                                          CTEMEK                                                       41w7             ------------         ----122(62.9%)237 35 111a.1L78149      41w15*                                                                       118230 38  91a.1L78149                                                          41w19         -----G------           ----  5( 2.6%) 10  2  21a.2L78156        41w20            --G--------A         ----  0  6  0  11a.3L78157                                                                       41w30                                                                          --------G---                                                                       ----                                                                  1( 0.5%)  8  6                                                                11a.4L78154                                                                     41m21                                                                       ------T--------                                                                      --L--                                                                  18( 9.4%) 37  7                                                                21a.5L78136                                                                    41m11                                                                          --GT--------                                                                       -L--  0                                                                0  0  11a.6L781                                                              40                     41m24            ---T-----G--         -L-E 12( 6.3%)  1  2  11a.7L78144       41m13            ---C--------         -L-- 14( 7.3%) 21  3  11a.8L78139       41m12            --GC--------         -L--  0  1  0  11a.9L78155                                                                       41m27                                                                          ---C-----G--                                                                       -L-E                                                                  3( 1.6%)  0  1                                                                1a.10L78137                                                                    total175(91.6%)                                                               95.1%100%          __________________________________________________________________________                                                              88%                 probe Codon 68-72           n=354/m=32                                                                              n=306                                                                              n=52 n=25                          __________________________________________________________________________          AGTACTAAATGGAGA                                                                             STKWR                                                        70w1                                                                        224(63.3%)230 39 131b.1,2L78147                                                70w8*                                                                        208210 38 111b.2L78144                                                         70m12                                                                        G-------                                                                      R-- 37(10.5%) 46  6  41b.3L78148                                               70m13                                                                        A-G-------                                                                    R--  0  0  1  21b.4L78133                                                       70w2                                                                        GA----------                                                                  D--- 25( 7.1%)  4  4  21b.5L78136                                               70m3          GA--G-------        DR-- 10( 2.8%)  3  1  01b.6pending                                                                  70m14               A--G----                                                                      NR-  7( 2.0%)  4  5  21b.7L78154                                               70m16                                                                        G---G----                                                                     AR-  2( 0.6%)  0  0  11b.8L78150                                               total305(86.2%) 91.8% 94.2% 96%                                             __________________________________________________________________________    probe Codon 72-77           n=364/m=20                                                                              n=306                                                                              n=52 n=25                          __________________________________________________________________________          AGAAAATTAGTAGATTTC                                                                          RKLVDF                                                       74w5                                                                        320(87.9%)264 48 161c.1L78150                                                   74w8          -----------C---        -----  9( 2.5%) 34  1  21c.2L78147       74w9                                                                        G-----G---                                                                      0 17  3  21c.3L78137                                                          74m6                                                                        G--------                                                                     V--  0  5  0  31c.4L78149                                                      74m12          ------AC----           --T--  0   1  1  11c.5L78136                                                                     total329(90.4%)                                                               93.5% 98.1%        __________________________________________________________________________                                                              96%                 probe Codon 182-185         n=322/m=12                                                                              n=306                                                                              n=52 n=25                          __________________________________________________________________________          CAATACATGGAT  QYMD                                                        184w11                                                                       285(88.5%)267 46 181d.1L78147                                                  184w17                                                                       G---------                                                                     16( 5.0%)  9  4  31d.2L78137                                                  184w21                                                                       T------                                                                         6( 1.9%)  4  2  11d.3L78145                                                  184w24                                                                       C                                                                               0  1  0  11d.4L78144                                                         184m12                                                                       G-----                                                                        V-  1( 0.3%)  8  0  11d.5L78142                                                184m28                                                                       A---                                                                          I-  3( 0.9%)  0  0  11d.6L78148                                                total311(96.6%) 93.8% 98.1%100%                                             __________________________________________________________________________    probe Codon 212-218         n=321/m=36                                                                              n=306                                                                              n=52 n=25                          __________________________________________________________________________          TGGGGATTTACCACACCAGAC                                                                       WGFTTPD                                                     215w11           G------------            ----  9( 2.8%) 15  3                                                                        21e.1L78146                                                                     215w9                                                                       ---------------                                                               -----142(44.2%)1                                                              78 24  31e.2L781                                                              41                    215w29.sup.f                                                                 142105 16  31e.2L78141                                                         215w33         -----C------              ----  9( 2.8%)  8  4  11e.3L781                                                              54                    215w10            C-----------            L--- 25( 7.8%) 10  0                                                                        21e.4L78150                                                                    215w27.sup.f        C-----                                                                        L- 25 14  0  21e.4L78150                                                       215m50         --GC--------              -L--  0  0  0  11e.5L78145                                                                    215w53                                                                       -----A------                                                                            -L--                                                                1( 0.3%)  1  3                                                                11e.6L78138                                                                    215w22                                                                             --T-------                                                              --                                                                            ----  3( 0.9%)                                                                10  2  11e.7L781                                                              34                    215m17         ------TA----              --Y- 88(27.4%) 50 12  71e.8L781                                                              44                    215m14         --G---TA----              --Y- 24( 7.5%) 24  1  11e.9L781                                                              49                    215m49         --G---TAT---              --Y-  0  2  0  21e.10L78135                                                                   215m45                                                                          ---TT------                                                                           -F--                                                                1( 0.3%) 16  0                                                                11e.11L78135                                                                  215m13                                                                       ---C--TA----                                                                            -LY-                                                                0  0  0                                                                       21e.12L78155                                                                   total302(94.1%)                                                               92.8% 90.4%        __________________________________________________________________________                                                              96%                 probe Codon 217-220         n=204/m=12                                                                              n=34 n=52 n=26                          __________________________________________________________________________          CCAGACAAAAAA  PDKK                                                        219w2                                                                        179(87.7%) 26 42 181f.1L78144                                                  219m4                                                                        C-----                                                                        Q-  1( 0.5%)  2  4  21f.2L78135                                                219m7                                                                        TC-----                                                                       Q-  0  0  0  11f.3L78133                                                       219m9                                                                        G-----                                                                        E-  0   0  0  11f.4pending                                                     total179(88.2%) 82.4% 82.7% 84.6%                                           __________________________________________________________________________     n=: amount of sequences (database retrieved), or isolates tested              m=: amount of motifs corresponding with the n database sequences              * : redundant probes                                                          RP: reference panel                                                               The total percentage for European and US samples is not the sum of        probe reactivities,                                                               but a result of the complete interpretations for these codons.       

REFERENCES

Asseline U, Delarue M, Lancelot G, Toulme F, Thuong N (1984) Nucleicacid-binding molecules with high affinity and base sequence specificity:intercalating agents covalently linked to oligodeoxynucleotides. Proc.Natl. Acad. Sci. USA 81(11):3297-301.

Barany F. Genetic disease detection and DNA amplification using clonedthermostable ligase. Proc Natl Acad Sci USA 1991; 88: 189-193.

Bej A, Mahbubani M, Miller R, Di Cesare J, Haff L, Atlas R. Mutiplex PCRamplification and immobilized capture probes for detection of bacterialpathogens and indicators in water. Mol Cell Probes 1990; 4:353-365.

Boom R., Sol C. J. A., Salimans M. M. M., et al. Rapid and simple methodfor purification of nucleic acids. J Clin Microbiol 1990; 28: 495-503.

Compton J. Nucleic acid sequence-based amplification. Nature 1991; 350:91-92.

Duck P. Probe amplifier system based on chimeric cyclingoligonucleotides. Biotechniques 1990; 9: 142-147.

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Frenkel L. Wagner L, Atwood S, Cummins T, Dewhurst S. Specific,sensitive and rapid assay for human immunodeficiency virus type 1 polmutations asssociated with resistance to zidovudine and didanosine. J.Clin. Microbiology 1995; 33: 342-347.

Guatelli J, Whitfield K, Kwoh D, Barringer K, Richman D, Gengeras T.Isothermal, in vitro amplification of nucleic acids by a multienzymereaction modeled after retroviral replication. Proc Natl Acad Sci USA1990; 87: 1874-1878.

Iversen A. K. N., R. W. Shafer, K. Wehrly, M. A. Winters, J. I. Mullins,B. Chesebro, and T. Merigan. Multidrug-resistant human immunodeficiencyvirus type 1 strains resulting from combination antiretroviral therapy.J. Virol. 1996; 70:1086-1090.

Kwoh D, Davis G, Whitfield K, Chappelle H, Dimichele L, Gingeras T.Transcription-based amplification system and detection of amplifiedhuman immunodeficiency virus type 1 with a bead-based sandwichhybridization format. Proc Natl Acad Sci USA 1989; 86: 1173-1177.

Kwok S, Kellogg D, McKinney N, Spasic D, Goda L, Levenson C, Sinisky J.Effects of primer-template mismatches on the polymerase chain reaction:Human immunodeficiency views type 1 model studies. Nucl. Acids Res.1990; 18: 999.

Landgren U, Kaiser R, Sanders J, Hood L. A ligase-mediated genedetection technique. Science 1988; 241:1077-1080.

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Lomeli H, Tyagi S, Printchard C, Lisardi P, Kramer F. Quantitativeassays based on the use of replicatable hybridization probes. Clin Chem1989; 35: 1826-1831.

Matsukura M, Shinozuka K, Zon G, Mitsuya H, Reitz M, Cohen J, Broder S(1987) Phosphorothioate analogs of oligodeoxynucleotides: inhibitors ofreplication and cytopathic effects of human immunodeficiency virus.Proc. Natl. Acad. Sci. USA 84(21):7706-10.

Mellors J, Larder B, Schinazzi R. Mutations in HIV-1 reversetranscripatse and protease associated with drug resistance.International Antiviral News (1195); 3: 8-13.

Miller P, Yano J, Yano E, Carroll C, Jayaram K, Ts'o P (1979) Nonionicnucleic acid analogues. Synthesis and characterization ofdideoxyribonucleoside methylphosphonates. Biochemistry 18(23):5134-43.

Nielsen P, Egholm M, Berg R, Buchardt O (1991) Sequence-selectiverecognition of DNA by strand displacement with a thymine-substitutedpolyamide. Science 254(5037): 1497-500.

Nielsen P, Egholm M, Berg R, Buchardt O (1993) Sequence specificinhibition of DNA restriction enzyme cleavage by PNA. Nucleic-Acids-Res.21(2):197-200.

Saiki R, Walsh P, Levenson C, Erlich H. Genetic analysis of amplifiedDNA with immobilized sequence-specific oligonucleotide probes Proc NatlAcad Sci USA 1989; 86:6230-6234.

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Stuyver L, Rossau R, Wyseur A, et al. Typing of hepatitis C virusisolates and characterization of new subtypes using a line probe assay.J. Gen. Virol. 1993; 74: 1093-1102.

Wu D, Wallace B. The ligation amplification reaction(LAR)--amplification of specific DNA sequences using sequential roundsof template-dependent ligation. Genomics 1989; 4:560-569.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 164                                         - -  - - (2) INFORMATION FOR SEQ ID NO: 1:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #1:                           - - AGAAATGGAA AAGGA              - #                  - #                      - #    15                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO: 2:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #2:                           - - TGTACAGAAA TGGAA              - #                  - #                      - #    15                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 3:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #3:                           - - AAATGGAAAA GGAAG              - #                  - #                      - #    15                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 4:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #4:                           - - TACAGAGATG GAAA              - #                  - #                      - #     14                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 5:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #5:                           - - GTACAGAGAT GGAAA              - #                  - #                      - #    15                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 6:                                    - 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#pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #7:                           - - AGAAATGGAG AAGGA              - #                  - #                      - #    15                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 8:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - 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-    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #9:                           - - GTACAGAGAT GGAA              - #                  - #                      - #     14                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 10:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #10:                          - - CAGAGATGGA AAAG              - #                  - #                      - #     14                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 11:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #11:                          - - AGAAATGGAA AAAGA              - #                  - #                      - #    15                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 12:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #12:                          - - GAAATGGAAA AAGA              - #                  - #                      - #     14                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 13:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #13:                          - - CAGAAATGGA AAAAGA             - #                  - #                      - #    16                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 14:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #14:                          - - AGAAATGGAA AAAGAA             - #                  - #                      - #    16                                                                   - 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#pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #17:                          - - AGAGCTGGAA AAGG              - #                  - #                      - #     14                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 18:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - 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-    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #19:                          - - GAGCTGGAAA AGG              - #                  - #                      - #      13                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 20:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #20:                          - - ACAGAATTGG AAAAG              - #                  - #                      - #    15                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 21:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #21:                          - - ACAGAATTGG AAAA              - #                  - #                      - #     14                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 22:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #22:                          - - ACAGAACTGG AAAA              - #                  - #                      - #     14                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 23:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #23:                          - - AGAATTGGAA GAGG              - #                  - #                      - #     14                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 24:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #24:                          - - CAGAATTGGA AGAGG              - #                  - #                      - #    15                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 25:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #25:                          - - AGAATTGGAA GAGGA              - #                  - #                      - #    15                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 26:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #26:                          - - AGAACTGGAA GAGG              - #                  - #                      - #     14                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 27:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #27:                          - - CAGAACTGGA AGAGG              - #                  - #                      - #    15                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 28:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #28:                          - - AGAACTGGAA GAGGA              - #                  - #                      - #    15                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 29:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #29:                          - - CAAAAATTGG GCCT              - #                  - #                      - #     14                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 30:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #30:                          - - ATTTCAAGAA TTGGG              - #                  - #                      - #    15                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 31:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #31:                          - - TTCAAAAGTT GGGC              - #                  - #                      - #     14                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 32:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #32:                          - - CAAAAATCGG GCCTG              - #                  - #                      - #    15                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 33:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #33:                          - - AAAAATCGGG CCTGA              - #                  - #                      - #    15                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 34:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #34:                          - - AAAGAAGAAA GACAG              - #                  - #                      - #    15                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 35:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #35:                          - - ATAAAGAAAA AGAACAGTA             - #                  - #                      - # 19                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 36:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #36:                          - - AGTACTAAAT GGAGAA             - #                  - #                      - #    16                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 37:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #37:                          - - AGTGATAAAT GGAGAA             - #                  - #                      - #    16                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 38:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #38:                          - - ACAGTACTAA ATGGAG             - #                  - #                      - #    16                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 39:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #39:                          - - ATCAGGATGG AGTTCATAAC CCATCCA          - #                  - #                 27                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 40:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #40:                          - - TAAATGGAGA AAATAG             - #                  - #                      - #    16                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 41:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #41:                          - - GTGATAGATG GAGAA              - #                  - #                      - #    15                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 42:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #42:                          - - GTACTAGATG GAGA              - #                  - #                      - #     14                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 43:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #43:                          - - AGTACTAGAT GGAGA              - #                  - #                      - #    15                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 44:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #44:                          - - AGTACAAGAT GGAGA              - #                  - #                      - #    15                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 45:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #45:                          - - CAGTAATAGA TGGAG              - #                  - #                      - #    15                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 46:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #46:                          - - ACAGTGCTAG ATGGA              - #                  - #                      - #    15                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 47:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #47:                          - - CAGTGCTAGA TGGA              - #                  - #                      - #     14                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 48:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #48:                          - - CAGTGCTAGA TGGA              - #                  - #                      - #     14                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 49:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #49:                          - - CAGTGATAGA TGGA              - #                  - #                      - #     14                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 50:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #50:                          - - CAGTGATAGA TGGAG              - #                  - #                      - #    15                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 51:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #51:                          - - AGTGATAGAT GGAG              - #                  - #                      - #     14                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 52:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #52:                          - - AGTGATAGAT GGAGA              - #                  - #                      - #    15                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 53:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #53:                          - - GAGAAAATTA GTAGATTT             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 54:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #54:                          - - AAAATTAGTA GACTTC             - #                  - #                      - #    16                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 55:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #55:                          - - GAGAAAGTTA GTGGATT             - #                  - #                      - #   17                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 56:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #56:                          - - AGAAAAGTAG TAGATTT             - #                  - #                      - #   17                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 57:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #57:                          - - AAAATTAACA GATTTC             - #                  - #                      - #    16                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 58:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #58:                          - - GAAAATTAAC AGATTT             - #                  - #                      - #    16                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 59:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #59:                          - - GAAAATTAAC AGATTTC             - #                  - #                      - #   17                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 60:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #60:                          - - CTTCCACAGG GATGG              - #                  - #                      - #    15                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 61:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #61:                          - - CTTCCACAAG GATGG              - #                  - #                      - #    15                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 62:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #62:                          - - TGCTCCCACA GGGATG             - #                  - #                      - #    16                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 63:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #63:                          - - CTTCCAATGG GATGG              - #                  - #                      - #    15                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 64:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #64:                          - - GCTTCCAATG GGATGG             - #                  - #                      - #    16                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 65:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #65:                          - - AGTTATCTAT CAATACAG             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 66:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #66:                          - - AGTTATCTGT CAATAC             - #                  - #                      - #    16                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 67:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #67:                          - - TCAATACATG GATGAGG             - #                  - #                      - #   17                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 68:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #68:                          - - TCAGTACATG GATGAGG             - #                  - #                      - #   17                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 69:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #69:                          - - ATCAATACAT GGATGA             - #                  - #                      - #    16                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 70:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #70:                          - - TCAGTACATG GATG              - #                  - #                      - #     14                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 71:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #71:                          - - ATCAATATAT GGATG              - #                  - #                      - #    15                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 72:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #72:                          - - ATCAATATAT GGATGA             - #                  - #                      - #    16                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 73:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #73:                          - - TCAATATATG GATGA              - #                  - #                      - #    15                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 74:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #74:                          - - TCAATACATG GACGA              - #                  - #                      - #    15                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 75:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #75:                          - - CAATACATGG ACGAT              - #                  - #                      - #    15                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 76:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #76:                          - - TCAATACATG GACGAT             - #                  - #                      - #    16                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 77:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #77:                          - - CAATACGTGG ATGAGGG             - #                  - #                      - #   17                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 78:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #78:                          - - AATACATAGA TGAT              - #                  - #                      - #     14                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 79:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #79:                          - - CAATACATAG ATGAT              - #                  - #                      - #    15                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 80:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #80:                          - - CAATACATAG ATGATT             - #                  - #                      - #    16                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 81:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #81:                          - - CAATACGTAG ATGAT              - #                  - #                      - #    15                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 82:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #82:                          - - TCAATACGTG GATGA              - #                  - #                      - #    15                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 83:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #83:                          - - TCAATACATA GATGAT             - #                  - #                      - #    16                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 84:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #84:                          - - ATCAATACAT AGATGAT             - #                  - #                      - #   17                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 85:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #85:                          - - GGATTTACCA CACCA              - #                  - #                      - #    15                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 86:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #86:                          - - GACTTACCAC ACCA              - #                  - #                      - #     14                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 87:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #87:                          - - GGTTTACCAC ACCA              - #                  - #                      - #     14                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 88:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #88:                          - - GATTTACCAC ACCA              - #                  - #                      - #     14                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 89:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #89:                          - - TTACTACACC AGAC              - #                  - #                      - #     14                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 90:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #90:                          - - TTACCACACC AGA              - #                  - #                      - #      13                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 91:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #91:                          - - TGGGGACTTA CCAC              - #                  - #                      - #     14                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 92:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #92:                          - - TGGGGATTTA CCAC              - #                  - #                      - #     14                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 93:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #93:                          - - GGGGTTCACC ACAC              - #                  - #                      - #     14                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 94:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #94:                          - - GGGATTTACC ACACCAG             - #                  - #                      - #   17                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 95:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #95:                          - - GGGATTTACC ACACCAG             - #                  - #                      - #   17                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 96:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #96:                          - - TGGGGACTTA CCACACC             - #                  - #                      - #   17                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 97:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #97:                          - - TGGGGGTTTA CCACACC             - #                  - #                      - #   17                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 98:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #98:                          - - GGGATTTACT ACACCAG             - #                  - #                      - #   17                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 99:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #99:                          - - GGGATTAACC ACAC              - #                  - #                      - #     14                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 100:                                  - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #100:                         - - GGGGATTAAC CACA              - #                  - #                      - #     14                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 101:                                  - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #101:                         - - TGGGGATTAA CCACA              - #                  - #                      - #    15                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 102:                                  - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #102:                         - - GGGGGTTAAC CACA              - #                  - #                      - #     14                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 103:                                  - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #103:                         - - GGGGTTAACC ACAC              - #                  - #                      - #     14                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 104:                                  - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #104:                         - - TGGGGGTTAA CCAC              - #                  - #                      - #     14                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 105:                                  - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #105:                         - - GGGATTGACC ACAC              - #                  - #                      - #     14                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 106:                                  - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #106:                         - - GGATTGACCA CACC              - #                  - #                      - #     14                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 107:                                  - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #107:                         - - GGGATTGACC ACA              - #                  - #                      - #      13                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 108:                                  - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #108:                         - - GGGACTGACC ACA              - #                  - #                      - #      13                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 109:                                  - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #109:                         - - GGGACTGACC ACAC              - #                  - #                      - #     14                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 110:                                  - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #110:                         - - TGGGGGTTAA CCACA              - #                  - #                      - #    15                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 111:                                  - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #111:                         - - TGTGGTTAAC CCCCA              - #                  - #                      - #    15                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 112:                                  - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #112:                         - - GGGGCTTACC ACAC              - #                  - #                      - #     14                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 113:                                  - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #113:                         - - GGACTTTACA CACC              - #                  - #                      - #     14                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 114:                                  - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #114:                         - - GGGTTTTACA CACC              - #                  - #                      - #     14                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 115:                                  - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #115:                         - - GGATTTTTCA CACCA              - #                  - #                      - #    15                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 116:                                  - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #116:                         - - GGATTTTACA CACC              - #                  - #                      - #     14                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 117:                                  - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #117:                         - - GGGATTTTAC ACACCAG             - #                  - #                      - #   17                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 118:                                  - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #118:                         - - GGGATTTTTC ACACCAG             - #                  - #                      - #   17                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 119:                                  - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #119:                         - - GGGATTTTAC ACAC              - #                  - #                      - #     14                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 120:                                  - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #120:                         - - GGGGATTTTA CACA              - #                  - #                      - #     14                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 121:                                  - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #121:                         - - CCCTAAAATG TGTG              - #                  - #                      - #     14                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 122:                                  - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #122:                         - - GGATTTTTCA CACC              - #                  - #                      - #     14                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 123:                                  - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #123:                         - - GATTTTTCAC ACCA              - #                  - #                      - #     14                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 124:                                  - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #124:                         - - GGGATTTTTC ACAC              - #                  - #                      - #     14                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 125:                                  - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #125:                         - - CCCCTAAAAT GTGT              - #                  - #                      - #     14                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 126:                                  - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #126:                         - - GGTTTTATAC ACCA              - #                  - #                      - #     14                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 127:                                  - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #127:                         - - GGGTTTTATA CACC              - #                  - #                      - #     14                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 128:                                  - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #128:                         - - GGGGTTTTAT ACAC              - #                  - #                      - #     14                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 129:                                  - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #129:                         - - GGGGGCTTAC CACA              - #                  - #                      - #     14                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 130:                                  - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #130:                         - - GGATTCTACA CACC              - #                  - #                      - #     14                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 131:                                  - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #131:                         - - GATTCTACAC ACC              - #                  - #                      - #      13                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 132:                                  - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #132:                         - - GGATTCTACA CAC              - #                  - #                      - #      13                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 133:                                  - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #133:                         - - GGGATTCTAC ACAC              - #                  - #                      - #     14                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 134:                                  - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #134:                         - - GGGTTTTATA CCCC              - #                  - #                      - #     14                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 135:                                  - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #135:                         - - GGTTTTATAC CCC              - #                  - #                      - #      13                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 136:                                  - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #136:                         - - GTTTTATACC CCA              - #                  - #                      - #      13                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 137:                                  - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #137:                         - - ACCAGACAAA AAACA              - #                  - #                      - #    15                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 138:                                  - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #138:                         - - GGGACTGACC ACAC              - #                  - #                      - #     14                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 139:                                  - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #139:                         - - CACCAGACAA AAAAC              - #                  - #                      - #    15                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 140:                                  - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #140:                         - - CAGACAAGAA ACAT              - #                  - #                      - #     14                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 141:                                  - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #141:                         - - CCAGACAAGA AACA              - #                  - #                      - #     14                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 142:                                  - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #142:                         - - ACCAGACAAG AAACA              - #                  - #                      - #    15                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 143:                                  - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #143:                         - - AGACAAAAAG CATC              - #                  - #                      - #     14                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 144:                                  - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #144:                         - - CAGACAAAAA GCAT              - #                  - #                      - #     14                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 145:                                  - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #145:                         - - CAGACAAAAA GCATC              - #                  - #                      - #    15                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 146:                                  - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #146:                         - - CCAGATAAAA AACA              - #                  - #                      - #     14                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 147:                                  - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #147:                         - - ACCAGATAAA AAAC              - #                  - #                      - #     14                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 148:                                  - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #148:                         - - CCCAGATAAA AAACA              - #                  - #                      - #    15                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 149:                                  - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #149:                         - - CCAGATAAAA AACATC             - #                  - #                      - #    16                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 150:                                  - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #150:                         - - CACCAGATAA AAAAC              - #                  - #                      - #    15                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 151:                                  - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #151:                         - - CAGACAAGAA ACATC              - #                  - #                      - #    15                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 152:                                  - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #152:                         - - ACCAGACAAG AAAC              - #                  - #                      - #     14                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 153:                                  - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #153:                         - - ACCAGACCAA AAACA              - #                  - #                      - #    15                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 154:                                  - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #154:                         - - ACCAGACGAA AAACA              - #                  - #                      - #    15                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 155:                                  - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #155:                         - - ACCAGATCAA AAACA              - #                  - #                      - #    15                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 156:                                  - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #156:                         - - ACCAGATCAA AAAC              - #                  - #                      - #     14                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 157:                                  - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #157:                         - - CACCAGATCA AAAAC              - #                  - #                      - #    15                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 158:                                  - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #158:                         - - ACCAGACGAA AAAC              - #                  - #                      - #     14                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 159:                                  - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #159:                         - - CCAGACGAAA AACA              - #                  - #                      - #     14                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 160:                                  - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #160:                         - - CCAGACCAAA AACA              - #                  - #                      - #     14                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 161:                                  - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #161:                         - - ACCAGACCAA AAAC              - #                  - #                      - #     14                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 162:                                  - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #162:                         - - GTACAGTATT AGTAGGACCT ACACCTGTC         - #                  - #                29                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 163:                                  - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #163:                         - - CCAAAAGTTA AACAATGGCC ATTGACAGA         - #                  - #                29                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 164:                                  - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #164:                         - - AGTTCATAAC CCATCCAAAG            - #                  - #                      - # 20                                                                 __________________________________________________________________________

What is claimed is:
 1. Method for determining the susceptibility toantiviral drugs of viruses which contain reverse transcriptase genes andare present in a biological sample, comprising:(i) hybridizingpolynucleic acids present in the sample with at least two reversetranscriptase (RT) gene probes, with said probes being applied to knownlocations on a solid support and with said probes being capable ofsimultaneously hybridizing to their respective target regions underappropriate hybridization and wash conditions allowing the detection ofhomologous targets, or with said probes hybridizing specifically with asequence complementary to any of said target sequences, or a sequencewherein T of said target sequence is replaced by U; (ii) detecting thehybrids formed in step (i); and (iii) inferring the nucleotide sequenceat the codons of interest as represented in any of FIG. 1, or Table 4and/or the amino acids of the codons of interest and/or antiviral drugresistance spectrum from the differential hybridization signal(s)obtained in step (ii);wherein said viruses are HIV strains, and whereinsaid RT gene probes are each selected from the group consisting of SEQID NO: 5, 6, 7, 20, 16, 23, 18, 17, 26, 44, 37, 41, 45, 47, 54, 55, 56,59, 67, 68, 71, 74, 77, 84, 87, 85, 92, 94, 91, 129, 100, 89, 114, 128,138, 156, 61, 62, 63, 64, 65, and
 66. 2. Method according to claim 1,wherein step (i) consists of hybridizing with at least two probes eachselected from the group consisting of SEQ ID NO: 5, 6, 7, 20, 16, 23,18, 17, and
 26. 3. Method according to claim 1, wherein step (i)consists of hybridizing with at least two probes each selected from thegroup consisting of SEQ ID NO: 44, 37, 41, 45, and
 47. 4. Methodaccording to claim 1, wherein step (i) consists of hybridizing with atleast two probes each selected from the group consisting of SEQ ID NO:54, 55, 56, and
 59. 5. Method according to claim 1, wherein step (i)consists of hybridizing with at least two probes each selected from thegroup consisting of SEQ ID NO: 67, 68, 71, 74, 77, and
 84. 6. Methodaccording to claim 1, wherein step (i) consists of hybridizing with atleast two probes each selected from the group consisting of SEQ ID NO:87, 85, 92, 94, 91, 129, 100, 89, 114, and
 128. 7. Method according toclaim 1, wherein step (i) consists of hybridizing with the probesconsisting of SEQ ID NO: 138 and
 156. 8. At least two probes on a solidsupport;wherein the probes comprise at least one first probe selectedfrom the group consisting of SEQ ID NO: 5, 6, 7, 20, 16, 23, 18, 17, 26,44, 37, 41, 45, 47, 54, 55, 56, 59, 67, 68, 71, 74, 77, 84, 87, 85, 92,94, 91, 129, 100, 89, 114, 128, 138, 156, 61, 62, 63, 64, 65, and 66 andat least one other second probe selected from the group consisting ofSEQ ID NO: 5, 6, 7, 20, 16, 23, 18, 17, 26, 44, 37, 41, 45, 47, 54, 55,56, 59, 67, 68, 71, 74, 77, 84, 87, 85, 92, 94, 91, 129, 100, 89, 114,128, 138, 156, 61, 62, 63, 64, 65, and
 66. 9. Composition comprising atleast one first probe selected from the group consisting of SEQ ID NO:5, 6, 7, 20, 16, 23, 18, 17, 26, 44, 37, 41, 45, 47, 54, 55, 56, 59, 67,68, 71, 74, 77, 84, 87, 85, 92, 94, 91, 129, 100, 89, 114, 128, 138,156, 61, 62, 63, 64, 65, and 66 and at least one other second probeselected from the group consisting of SEQ ID NO: 5, 6, 7, 20, 16, 23,18, 17, 26, 44, 37, 41, 45, 47, 54, 55, 56, 59, 67, 68, 71, 74, 77, 84,87, 85, 92, 94, 91, 129, 100, 89, 114, 128, 138, 156, 61, 62, 63, 64,65, and 66.